Process of delivering small rnas to sperm

ABSTRACT

Methods and compositions directed to altering a population of sRNAs in a sperm using vesicles isolated from an epididymosome are provided. Methods and compositions directed to altering a population of sRNAs in an oocyte using vesicles isolated from an epididymosome are also provided. Methods for altering an sRNA population in a sperm or an oocyte can be used to prevent, or reduce the severity of, a disease, disorder, or condition that would otherwise be inherited by progeny. For example, certain epigenetic inherited conditions due to paternal effects, such as certain metabolic and stress disorders and conditions, can be ameliorated in progeny using sperm or oocytes having an altered sRNA population.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.16/315,004, filed Jan. 3, 2019, which is a 35 U.S.C. § 371 filing ofInternational Patent Application No. PCT/US2017/041647, filed Jul. 12,2017, which claims priority to U.S. Provisional Patent Application Ser.No. 62/363,174, filed Jul. 15, 2016, the contents of which areincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.ES025458 and HD080224 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

TECHNICAL FIELD

The disclosed methods and compositions are directed to the field ofreproductive biology. Specifically, the disclosed methods andcompositions are directed to the delivery of small RNAs to sperm toeffectuate changes in sperm RNA content.

BACKGROUND

Mendelian laws of genetics govern most inheritance, and most epigeneticmodifications an organism may acquire are reset between generations.However, there has recently been growing evidence to supporttransgenerational epigenetic inheritance, where some epigeneticphenotypes are maintained through generations (Lim and Brunet, TrendsGenet. 2013 29(3):176-186).

For example, animal studies and human cohort studies have suggested thatmetabolic changes in parents can be inherited epigenetically byoffspring. In rats, a high fat diet in male parents affects glucosemetabolism in female offspring (Ng et al., Nature 2010; 467: 963-966).Overfeeding male mice also results in the observation of metabolicchanges in the two subsequent generations of male offspring (Pentinat etal., Endocrinology 2010; 151: 5617-5623). When female rats are fed ahigh fat diet, metabolism was also found to be altered in the next twogenerations, with only the females of the third generation showing thismetabolic alteration, this latter phenotype being passed only paternally(Dunn and Bale, Endocrinology 2009; 150: 4999-5009; Dunn and Bale,Endocrinology 2011; 152: 2228-2236). If male rat parents are fed a lowprotein diet, then the offspring show metabolic alterations, such aslowered liver cholesterol (Carone et al., Cell 2010; 143: 1084-1096).

Human cohort studies also have proven to be the source of strikingobservations regarding apparent epigenetic effects on metabolism. Forexample, when mothers are exposed to famine during pregnancy, metabolismin male offspring is affected (Lumey et al., Am. J. Clin. Nutr. 2009;89: 1737-1743). Second generation offspring also demonstrate alterationsin metabolism, including a predisposition to suffer metabolic disease(Painter et al., Bjog. 2008; 115: 1243-1249). In another study, low foodintake during adolescence correlated with an increase in survival ofgrandchildren (Pembrey et al., Eur. J. Hum. Genet. 2006; 14: 159-166).

Another example of intergenerational transmission of environmentalinformation is the effect of stress experienced by parents, whichappears to affect stress-related behaviors, and glucose metabolism, inoffspring. For example, when parental male mice were exposed to maternalseparation and unpredictable maternal stress (MSUS), depressive-likebehaviors were observed in two subsequent generations (Franklin et al.,Biol. Psychiatry 2010; 68: 408-415; see also Gapp, K et al., NatureNeuroscience 2014; 17(5): 667-669).

The mechanisms responsible for epigenetic inheritance patterns are justbeginning to be understood. Mechanisms that have been implicated inthese inheritance patterns thus far include histone modifications, DNAmethylation, and non-coding RNAs, including RNA interference (RNAi)machinery, small interfering RNAs (siRNAs), Piwi-interacting RNAs(piRNAs) and microRNAs (miRNAs) (Lim and Brunet, Trends Genet. 201329(3):176-186). For example, there is evidence that paternal dietaryconditions that affect offspring metabolism also affect the sperm smallRNA payload (Sharma et al., Science 2016; 351(6271): 391-396). Ifpurified sperm RNAs are injected into naive one-cell embryos,alterations in metabolism are observed in the resultant offspring(Grandjean et al., Sci Rep 2015; 5:18193; see also Chen, Q et al.,Science 2016; 351: 397-400). Likewise, when total sperm RNA fromtraumatized males was injected into fertilized wild-type oocytes, theresultant offspring displayed metabolic changes (Gapp et al., NatureNeuroscience 2014; 17(5): 667-669). Finally, injecting ninesperm-specific miRNAs into zygotes that were identified in a paternalstress mouse model recapitulated the stress dysregulation phenotype inoffspring (Rodgers et al., PNAS 2015; 112(44): 13699-13704).

There is a need in the art to efficiently modify sperm RNA payload to,for example, decrease the transmission of disease or disorders.

SUMMARY

In a first aspect, disclosed herein is a method of altering a populationof sRNAs in a sperm of a subject, comprising contacting the sperm withan sRNA-containing vesicle isolated from an epididymosome to produce asperm having an altered sRNA population. In embodiments, the sRNA isselected from the group consisting of a siRNA, a miRNA, a piRNA, asnoRNA, a srRNA, a U-RNA, and a tRNA fragment. In further embodiments,the tRNA fragment is selected from the group consisting of atRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment,a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTTfragment, and a tRNA-His-GTG fragment. In an embodiment, theepididymosome is selected from the group consisting of caputepididymosome, corpus epididymosome, and cauda epididymosome.

In some embodiments, prior to contacting the sperm, the sperm isimmature and altering an sRNA population increases sperm maturity. Inother embodiments, prior to contacting the sperm, the sperm is defectiveand altering an sRNA population diminishes at least one defect. In suchembodiments, the defective sperm can comprise a defect selected from thegroup consisting of a reduced level of sRNA, at least one aberrant sRNA,or absence of at least one sRNA that is present in healthy mature sperm.In an embodiment, the defective sperm comprises a defect in siRNA,miRNA, piRNA, snoRNA, srRNA, U-RNA, and tRNA fragment content. Infurther embodiments, the tRNA fragment content comprises a defectselected from the group consisting of tRNA-Gly-CCC fragment content,tRNA-Gly-TCC fragment content, tRNA-Gly-GCC fragment content,tRNA-Val-CAC fragment content, tRNA-Glu-CTC fragment content,tRNA-Lys-CTT fragment content, and tRNA-His-GTG fragment content. Inother embodiments, the defective sperm comprises a decrease in at leastone let-7 species of RNA when compared to a healthy sperm.

In certain embodiments, after altering the RNA content, the sperm isused to fertilize an oocyte.

In an embodiment, the subject is a mammal, such as a primate, such as ahuman.

In certain embodiments, the sperm that is altered is obtained from thesubject's caput epididymis, corpus epididymis, cauda epididymis, vasdeferens, testis, or ejaculate. In further embodiments, the sperm isobtained from the subject's caput epididymis, corpus epididymis, orcauda epididymis using microscopic or microsurgical epididymal spermaspiration (MESA) or percutaneous epididymal sperm aspiration (PESA). Inyet other further embodiments, the sperm is obtained from the subject'stestis using a technique selected from the group consisting of needleaspiration (TESA), percutaneous or open surgical biopsy (TESE),multibiopsy TESE, microdissection TESE, site-directed TESE after fineneedle aspiration mapping, and MicroTESE. Such techniques are routinelyused in assisted reproduction.

In certain embodiments, the subject whose sperm is altered isexperiencing a condition selected from the group consisting of astress-related disease or disorder, a dietary restriction, and obesity.In an embodiment, the dietary restriction is protein deficiency. Inanother embodiment, the stress-related disease or disorder is selectedfrom the group consisting of major depressive disorder, dysthymia,bipolar disorder, generalized anxiety disorder, a phobia, social anxietydisorder, separation anxiety disorder, agoraphobia, and panic disorder.

In embodiments, the vesicle that is contacted to the sperm isheterologous to the subject. In other embodiments, the vesicle isautologous to the subject. In yet other embodiments, the vesiclecomprises a heterologous RNA; the heterologous RNA can comprise a smallRNA (sRNA). Such sRNA can be one selected from the group consisting of asiRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNA fragment.In embodiments where the sRNA is a tRNA fragment, the tRNA fragment canbe selected from the group consisting of a tRNA-Gly-CCC fragment, atRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment,a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTGfragment. In an embodiment where the sRNA is an miRNA, it can beselected from the group consisting of miR-10a/b, miR-141, miR-143,miR-148 and miR-200a. In other embodiments, the vesicle comprisesautologous RNA. Such a vesicle can comprise sRNA that can be selectedfrom the group consisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA,a U-RNA, and a tRNA fragment. In the case where the sRNA is a tRNAfragment, the tRNA fragment can be selected from the group consisting ofa tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCCfragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, atRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In an embodimentwhere the sRNA is an miRNA, it can be selected from the group consistingof miR-10a/b, miR-141, miR-143, miR-148 and miR-200a. In otherembodiments, the vesicle comprises an artificial (synthetic) RNA. Insuch vesicles, the sRNA can be selected from the group consisting of asiRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNA fragment.In the case where the sRNA is a tRNA fragment, then the tRNA fragmentcan be selected from the group consisting of a tRNA-Gly-CCC fragment, atRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment,a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTGfragment. In an embodiment where the sRNA is an miRNA, it can beselected from the group consisting of miR-10a/b, miR-141, miR-143,miR-148 and miR-200a. In other embodiments, the vesicle comprises atransgene.

In certain embodiments, the altered sperm fertilizes an oocyte in vitro.In other embodiments, the sperm is used in intracytoplasmic sperminjection (ICSI). These embodiments can further comprise implanting thefertilized oocyte to a second subject (e.g., a non-human subject) toproduce a progeny.

In other embodiments, the altered sperm fertilizes an oocyte in vivo.

In embodiments, prior to contacting the sperm with a vesicle, the spermare frozen.

In a second aspect, disclosed herein is a method of treating anepigenetically inheritable trait at risk of being transmitted to aprogeny of a subject, comprising altering a population of sRNAs in asperm from the subject by contacting the sperm with an sRNA-containingvesicle isolated from an epididymosome and fertilizing an oocyte withthe sperm to produce the progeny. In embodiments, the sRNA is selectedfrom the group consisting of a siRNA, a miRNA, a piRNA, a snoRNA, asrRNA, a U-RNA, and a tRNA fragment. In embodiments where the sRNA is atRNA fragment, the tRNA fragment can be selected from the groupconsisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, atRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment,a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In embodimentswhere the sRNA is an miRNA, it can be selected from the group consistingof miR-10a/b, miR-141, miR-143, miR-148 and miR-200a. In otherembodiments, the vesicle comprises a transgene.

In embodiments, the epididymosome is selected from the group consistingof caput epididymosome, corpus epididymosome, and cauda epididymosome.

In embodiments, the epigenetically inheritable trait is a disease ordisorder that is a metabolic or stress-related disease or disorder. Insome embodiments, the metabolic disease or disorder comprises a glucoseor hepatic metabolic disease or disorder. In further embodiments, thehepatic metabolic disease or disorder comprises reduced sterolbiosynthesis. In yet further embodiments, the reduced sterolbiosynthesis comprises reduced cholesterol biosynthesis. In even furtherembodiments, hepatic Sqle gene expression is upregulated. In otherembodiments, the stress-related disease or disorder is selected from thegroup consisting of major depressive disorder, dysthymia, bipolardisorder, generalized anxiety disorder, a phobia, social anxietydisorder, separation anxiety disorder, agoraphobia, and panic disorder.

In embodiments, the progeny lacks symptoms of the epigeneticallyinheritable trait. In other embodiments, the progeny has amelioratedsymptoms of the epigenetically inheritable trait.

In embodiments of this second aspect, the sperm comprises a defectselected from the group consisting of a reduced level of sRNA, at leastone aberrant sRNA, or absence of at least one sRNA that is present inhealthy mature sperm. In some embodiments, the sperm comprises a defectin siRNA, miRNA, piRNA, snoRNA, srRNA, U-RNA, and tRNA fragment content.In those embodiments wherein the defect is in tRNA content, the defectcan be selected from the group consisting of tRNA-Gly-CCC fragmentcontent, tRNA-Gly-TCC fragment content, tRNA-Gly-GCC fragment content,tRNA-Val-CAC fragment content, tRNA-Glu-CTC fragment content,tRNA-Lys-CTT fragment content, and tRNA-His-GTG fragment content. Inother embodiments, prior to contacting the sperm, the sperm comprises adecrease in at least one let-7 species of RNA when compared to a healthysperm.

In embodiments, the subject is a mammal, such as a primate, such as ahuman.

In embodiments, the sperm is obtained from the subject's caputepididymis, corpus epididymis, cauda epididymis, vas deferens, testis,or ejaculate. In such embodiments where the sperm obtained from thesubject's caput epididymis, corpus epididymis, or cauda epididymis,microscopic or microsurgical epididymal sperm aspiration (MESA) orpercutaneous epididymal sperm aspiration (PESA) is used. In otherembodiments wherein the sperm is obtained from the subject's testis, atechnique selected from the group consisting of needle aspiration(TESA), percutaneous or open surgical biopsy (TESE), multibiopsy TESE,microdissection TESE, site-directed TESE after fine needle aspirationmapping, and MicroTESE can be used. Such techniques are routinely usedin assisted reproduction.

In embodiments of this second aspect, the subject is experiencing acondition selected from the group consisting of a stress-related diseaseor disorder, dietary restriction, and obesity. In further embodiments,the dietary restriction is protein deficiency. In other furtherembodiments, the stress-related disease or disorder is selected from thegroup consisting of major depressive disorder, dysthymia, bipolardisorder, generalized anxiety disorder, a phobia, social anxietydisorder, separation anxiety disorder, agoraphobia, and panic disorder.

In some embodiments, the vesicle is heterologous to the subject. Inother embodiments, the vesicle is autologous to the subject. In otherembodiments, the vesicle comprises a heterologous RNA. In furtherembodiments, the heterologous RNA comprises a small RNA (sRNA). In suchembodiments, the sRNA can be selected from the group consisting of asiRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNA fragment.In embodiments where the sRNA is a tRNA fragment, the tRNA fragment canbe selected from the group consisting of a tRNA-Gly-CCC fragment, atRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment,a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTGfragment. In other embodiments, the vesicle comprises autologous RNA. Insuch vesicles, the sRNA can be selected from the group consisting of asiRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNA fragment.In those embodiments where the sRNA is a tRNA fragment, the tRNAfragment can be selected from the group consisting of a tRNA-Gly-CCCfragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, atRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment,and a tRNA-His-GTG fragment. In other embodiments, the vesicle comprisesan artificial (synthetic) RNA. In such vesicles, the sRNA can beselected from the group consisting of a siRNA, miRNA, a piRNA, a snoRNA,a srRNA, a U-RNA, and a tRNA fragment. In those embodiments where thesRNA is a tRNA fragment, the tRNA fragment can be selected from thegroup consisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, atRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment,a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In otherembodiments, the vesicle comprises a transgene.

In yet more embodiments of this second aspect, the sperm fertilizes anoocyte in vitro. In other embodiments, the sperm is used inintracytoplasmic sperm injection (ICSI).

Some embodiments comprise implanting the fertilized oocyte to a secondsubject to produce a progeny. In other embodiments, the sperm fertilizesan oocyte in vivo.

In embodiments, prior to contacting the sperm with an vesicle, the spermare frozen.

In a third aspect, disclosed herein are pharmaceutical compositionscomprising an vesicle comprising a small RNA molecule (sRNA). Inembodiments, the sRNA is selected from the group consisting of a siRNA,a miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNA fragment. Inthose embodiments where the sRNA is a tRNA fragment, the tRNA fragmentcan be selected from the group consisting of a tRNA-Gly-CCC fragment, atRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment,a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTGfragment.

In some embodiments, the pharmaceutical composition is a vaginal foam orgel.

In some embodiments, the vesicle is an exosome; in yet furtherembodiments, the exosome is an epididymosome. In further embodiments,the epididymosome is selected from the group consisting of caputepididymosome, corpus epididymosome, and cauda epididymosome. In otherembodiments, the vesicle is a seminosome or a prostasome. In otherembodiments, the vesicle is a microvesicle.

In embodiments of this third aspect, the vesicle comprises aheterologous RNA. In further embodiments, the heterologous RNA comprisesa small RNA (sRNA). In yet further embodiments, the sRNA is selectedfrom the group consisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA,a U-RNA, and a tRNA fragment. In those embodiments where the sRNA is atRNA fragment, the tRNA fragment can be selected from the groupconsisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, atRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment,a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In otherembodiments, the vesicle comprises autologous RNA. In such embodiments,the vesicle comprises an sRNA that can be selected from the groupconsisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and atRNA fragment. In those embodiments where the sRNA is a tRNA fragment,the tRNA fragment can be selected from the group consisting of atRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment,a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTTfragment, and a tRNA-His-GTG fragment. In other embodiments, the vesiclecomprises an artificial (synthetic) RNA. In such embodiments, thevesicle comprises an sRNA that can be selected from the group consistingof a siRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNAfragment. In those embodiments where the sRNA is a tRNA fragment, thetRNA fragment can be selected from the group consisting of atRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment,a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTTfragment, and a tRNA-His-GTG fragment. In an embodiment, the vesiclecomprises a transgene.

In certain embodiments of any of the aspects above in which the sRNA isan miRNA, it may be selected from the group consisting of miR-10a/b,miR-141, miR-143, miR-148 and miR-200a.

In a fourth aspect, disclosed herein is a method of altering the sRNApopulation in an oocyte, comprising altering a population of sRNA in asperm by contacting a sperm with a vesicle isolated from anepididymosome (e.g., a caput epididymosome, a corpus epididymosomeand/or a cauda epididymosome) to produce a sperm having an altered sRNApopulation, and fertilizing the oocyte with the sperm having an alteredsRNA population. In certain embodiments, the sRNA is selected from thegroup consisting of a siRNA, a miRNA, a piRNA, a snoRNA, a srRNA, aU-RNA, and a tRNA fragment. In certain embodiments, the tRNA fragment isselected from the group consisting of a tRNA-Gly-CCC fragment, atRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment,a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTGfragment.

In certain exemplary embodiments, the sperm comprises a defect selectedfrom the group consisting of a reduced level of sRNA, at least oneaberrant sRNA, or absence of at least one sRNA that is present inhealthy mature sperm. In certain exemplary embodiments, the spermcomprises a defect in siRNA, miRNA, piRNA, snoRNA, srRNA, U-RNA, andtRNA fragment content.

In certain embodiments, the tRNA fragment content comprises a defectselected from the group consisting of tRNA-Gly-CCC fragment content,tRNA-Gly-TCC fragment content, tRNA-Gly-GCC fragment content,tRNA-Val-CAC fragment content, tRNA-Glu-CTC fragment content,tRNA-Lys-CTT fragment content, and tRNA-His-GTG fragment content.

In certain embodiments, the miRNAs is selected from the group consistingof miR-10a/b, miR-141, miR-143, miR-148 and miR-200a. In certainembodiments, the vesicle comprises a synthetic RNA and/or a transgene.

In certain exemplary embodiments, the sperm fertilizes an oocyte invitro or in vivo. In other embodiments, the sperm is used inintracytoplasmic sperm injection (ICSI). In certain embodiments, themethod further includes the step of implanting the fertilized oocyteinto a second, non-human subject to produce a progeny. In certainembodiments, the sperm are frozen prior to contacting the vesicle.

In a fifth aspect, disclosed herein is a method of altering a populationof sRNAs in an isolated sperm, comprising contacting the isolated spermwith an sRNA-containing vesicle isolated from a caput epididymosome toproduce a sperm having an altered sRNA population.

In certain embodiments, the sperm having an altered sRNA populationexhibits an increase in the levels of miRNAs and/or tRNA fragmentscompared to the levels of miRNAs and/or tRNA fragments in the isolatedsperm prior to contacting the sRNA-containing vesicle. In otherembodiments, a tRNA fragment is selected from the group consisting of atRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment,a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTTfragment and a tRNA-His-GTG fragment. In still other embodiments, amiRNA is selected from the group consisting of miR-10a/b, miR-141,miR-143, miR-148 and miR-200a.

In certain embodiments, the levels of tRNA fragments present areincreased by at least about 5%, at least about 6%, at least about 7%, atleast about 8%, at least about 9% or at least about 10% compared tolevels of tRNA fragments in the isolated sperm prior to contacting withthe sRNA-containing vesicle. In other embodiments, the levels of tRNAfragments present are increased by at least about two-fold compared tolevels of tRNA fragments in the isolated sperm prior to contacting withthe sRNA-containing vesicle.

In certain embodiments, the caput epididymosome is between about 100 nmand about 400 nm in diameter, between about 250 nm and about 350 nm indiameter, between about 120 nm and about 170 nm in diameter, or about150 nm in diameter. In certain embodiments, the caput epididymosome isisolated from an epididymal sample and/or is isolated from theepididymal sample by ultracentrifugation.

In a fifth aspect, disclosed herein is a method of correcting adevelopmental defect in a zygote comprising microinjecting the zygotewith a tRNA-Gly-GCC fragment to correct the developmental defect. Incertain embodiments, the expression level of one or more genesassociated with zygote development is altered. In other embodiments, theexpression level of one or more genes associated with zygote developmentis downregulated.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings.

FIGS. 1A-1D shows the characterization of small RNAs in sperm. FIG. 1Ashows small RNA sequencing data from mature cauda sperm samples. Sizesof deep sequencing reads are shown for the average of 12 small (<40 nt)cauda sperm RNA datasets. FIGS. 1B-1D show examples of abundant tRNAfragments (tRFs) in cauda sperm. tRNA fragments from the 5′ end oftRNA-Gly-GCC (FIG. 1B), tRNA-Val-CAC (FIG. 1C), and tRNA-Glu-CTC (FIG.1D) are shown schematically, with arrows indicating dominant 3′ ends.

FIGS. 2A-2C show that tRNA fragments are abundant in the epididymis.FIG. 2A shows a Northern blot analysis of total RNA isolated fromtestis, cauda epididymis, and caput epididymis, as indicated. FIG. 2Bshows the quantitation of Northern blot data. Bars show levels of tRFsin testis, caput epididymis, and cauda epididymis, normalized to testislevels. Error bars show s.e.m. FIG. 2C shows pie charts showing thepercentage of small RNAs mapping to the indicated features, for eachtissue. rRNA-mapping reads are excluded.

FIGS. 3A-3B show that tRNA cleavage predominantly occurs downstream ofthe testis. FIG. 3A shows that sperm RNA payload diverges dramaticallyfrom the RNA population in testes. Small (<40 nt) RNA data from caudasperm and from testes were normalized to parts per million (ppm) totalreads (excluding rRNA-mapping reads), and data are shown for all RNAspresent at greater than 5 ppm in the sperm or the testis averageddatasets. Scatterplot shows abundance of small RNAs in testis (x axis,log 10 scale) vs. sperm (y axis, log 10 scale), with RNAs mapping totRNA genes, to microRNAs, to repeat elements/unique piRNAs, and to allother transcripts (fragments of mRNAs, snoRNAs, etc.) all indicatedseparately. FIG. 3B shows a schematic of murine epididymis. Spermexiting the testis first enter the proximal (caput) epididymis, thenproceed distally to the corpus and cauda epididymis, and exit via thevas deferens.

FIG. 4 shows a cartoon showing testicular spermatogenesis andpost-testicular maturation in the epididymis. For each purified gametepopulation, pie charts show the relative abundance of tRNA fragments,microRNAs, piRNAs (defined as reads mapping to either repeatmaskerconsensus sequences or to unique piRNA clusters), and to Refseq (mRNAfragments), as indicated. Data are shown for three separate fractions ofpurified testicular germ cells, and for sperm isolated from caput andcauda epididymis, as indicated. Consistent with the low levels of tRNAfragments found in intact testes, spermatocytes and two populations ofpost-meiotic spermatids carry extremely low levels of tRNA fragments,indicating that the absence of tRNA fragments in intact testis is not aresult of contamination by testicular somatic cells.

FIGS. 5A-5B show changes in sperm tRF payload during epididymal transit.FIG. 5A shows that tRNA fragments that are more abundant in cauda,relative to caput, epididymis are also gained in cauda sperm. Thescatterplot shows the relative changes between caput and caudaepididymis (x axis—positive values show cauda-enriched RNAs) compared torelative changes between caput and cauda sperm (y axis). Note that giventhe normalization to total small RNA abundance, a “loss” of a given tRFpresent in caput sperm could result from degradation of this tRF, orfrom constant abundance of this tRF in the face of overall tRF gain.FIG. 5B shows proximal-distal biases for tRFs in the epididymis and insperm samples, averaged for each anticodon. Only tRFs with an averageabundance of >100 ppm small RNAs are shown, and tRFs are ordered bycauda/caput ratio for epididymis samples.

FIGS. 6A-6C show the characterization of cauda epididymosomes. FIG. 6Ashow a transmission electron micrograph of purified caudaepididymosomes, showing abundant vesicles of about 120-150 nm. FIG. 6Bshows epididymosome size distribution. Nanosight sizing data for twoindependent cauda epididymosome preps. Data for 0-200 nm are shown inmain panel, while inset shows 0-500 nm zoom-in. FIG. 6C shows thatepididymosomal preparations are not contaminated with free RNA, or withfragments of sperm. tRNA fragments are protected from RNaseA treatment,indicating their presence in vesicles. In addition, epididymosomespurified from tdrdl−/− mice, which lack mature sperm, carry high levelsof tRNA fragments, indicating that our epididymosome preparations arenot simply fragments generated from maturing sperm such as the residualbody.

FIG. 7 shows a comparison of small RNA payloads of cauda sperm vs. caudaepididymosomes.

FIGS. 8A-8E show the characterization of caput epididymosomepreparations. FIG. 8A shows a schematic of murine epididymis. Circlesrepresent epididymosomes. FIG. 8B shows a transmission electronmicrograph of purified cauda epididymosomes (top panel), showingabundant vesicles of approximately 120-150 nm. Lower panel shows vesiclesize distributions for epididymosomes isolated from cauda or from caputepididymis, obtained using nanosight sizing. A subtle increase inapproximately 250-300 nm vesicles is apparent in cauda samples. FIG. 8Cshows distributions for epididymosomal small (<40 nt) RNA-Seq libraries,showing highly abundant 28-32 nt (approximately 87% of reads) 5′ tRNAfragments for cauda epididymosomes. In contrast, caput epididymosomesprimarily carry microRNAs, with approximately 28% of reads mapping to 5′tRFs. FIG. 8D shows that small RNA populations in epididymosomes arehighly correlated with those in mature sperm. The scatterplot showsabundance of various classes of small RNAs for sperm (x axis, log scale)and epididymosomes (y axis, log scale). FIG. 8E shows the consistentproximal-distal biases for specific RNAs in epididymis and epididymosomesamples. For all RNAs with a maximum abundance of greater than 10 ppm ineither caput or cauda samples, the log 2(cauda/caput) ratio is plottedfor epididymis (x axis) vs. epididymosomes (y axis).

FIGS. 9A and 9B show our ability to deliver small RNAs to immature spermvia fusion with epididymosomes. FIG. 9A shows Taqman analysis of theindicated tRFs in purified caput sperm and in “reconstituted” caputsperm which have been incubated with cauda epididymosomes, showing gainof tRFs relative to normalization control let-7. FIG. 9B shows theresults of deep sequencing of sperm reconstitutions. In each case, tRFsare aggregated by codon, and data are normalized to levels oftRF-Glu-CTC. For bull, data shown mean and s.e.m. for four replicatereconstitutions experiments. For mouse, deep sequencing libraries wereunder sequenced approximately 100-200 thousand reads), and only tworeplicates were sequenced, but the same trends seen between naturalcaput and cauda sperm were recapitulated, with cauda-enriched tRFs suchas tRF-Val-CAC being delivered to caput sperm via fusion with caudaepididymosomes.

FIGS. 10A-10F show that tRF-Gly-GCC regulates MERVL-driven transcriptsin the early embryo. FIG. 10A shows Affymetrix microarray data for mRNAabundance in embryonic stem cells transfected with an LNA antisenseoligo targeting tRF-Gly-GCC. The X axis shows abundance of mRNAs inanti-GFP knockdown cells, and the y axis shows mRNA abundance for cellstransfected with an LNA antisense targeting the 5′ end of tRF-Gly-GCC.Data represent average of seven replicates. FIG. 10B shows the effect oftRF-Gly-GCC knockdown on MERVL is isoacceptor-specific. Affymetrix datafor knockdown studies with the indicated LNA antisense oligos. Allcomparisons are to GFP siRNA transfections done in the same batch.Identical results are obtained when comparing to mock-transfected EScells. All genes showing abundance changes of 2-fold or greater in 2 ormore samples are shown. FIG. 10C shows RNA-Seq data for four pooledreplicate samples of ES cells transfected with shRNA against GFP, orwith the anti-tRF-Gly-GCC LNA oligo, as indicated.

FIG. 10D shows a schematic showing genomic context for four tRF-Gly-GCCtarget genes, showing MERVL LTRs associated with all target genes. Someadditional target genes, such as the Tdpoz cluster, are not as closelyassociated with MERVL LTRs, but instead are located in large MERVL-richgenomic clusters, and have also been shown to be part of theMERVL-regulated gene expression program (Macfarlan, T S, et al. 2012.Nature 487: 57-63). FIG. 10E shows that inhibition of tRF-Gly-GCCaffects MERVL target expression in 4-cell embryos. Control zygotes weregenerated via IVF, and then either mock-injected or injected with anantisense oligonucleotide targeting tRF-Gly-GCC. Embryos were thenallowed to develop to the 4-cell stage, and subject to single-embryoRNA-Seq. Averaged single embryo RNA-Seq data for control (n=28) ortRF-inhibited (n=27) embryos. Among genes upregulated at least 2-fold onaverage, those previously described as MERVL targets are indicatedseparately. FIG. 10F shows examples of single embryo data for two MERVLtargets. Here, each bar represents mRNA abundance from a single embryo,with embryos ordered from highest to lowest expression for eachcondition.

FIGS. 11A-F show paternal dietary effects on preimplantationdevelopment. FIG. 11A shows embryos generated by IVF that were culturedfor varying times, then subject to single embryo RNA-Seq. FIG. 11B showssingle-embryo data for preimplantation embryos represented via PCA:first two principal components explain 74% of dataset variance. FIG. 11Cshows mRNA abundance in 2-cell embryos generated via IVF using Controlvs. Low Protein sperm (n=41 C and 39 LP). Cumulative distribution plotsfor tRF-Gly-GCC targets (p=4.5×10-7, KS test), other MERVL targets (17)(p=2.5×10-13), and all remaining genes, showing percentage of genes withthe average Log 2(LP/C) indicated on the x axis. Low Protein embryosexhibit a significant shift to lower expression of MERVL targets. Bottompanels show individual embryo data for two targets. FIG. 11D shows smallRNAs isolated from Control or Low Protein cauda sperm were microinjectedinto control zygotes. RNA-Seq (n=42 C and 46 LP embryos) revealsdownregulation of tRF-Gly-GCC targets (p=4.8×10-14) driven by LowProtein RNA. FIG. 11E shows the effects of synthetic tRF-Gly-GCC on2-cell gene regulation, showing significant (p=0.0001) downregulation oftarget genes in embryos injected with tRF-Gly-GCC (n=26) vs. GFPcontrols (n=11). Inset shows effects of tRF-Glu-CTC (n=6). FIG. 11Fshows effects of epididymal passage on embryonic gene regulation. Intactsperm isolated from rete testis (n=12), or cauda epididymis (n=9), wereinjected into control oocytes, and mRNA abundance was analyzed as above.

FIGS. 12A-12H show paternal dietary effects on preimplantationdevelopment. FIG. 12A shows subjected cumulative distribution plot forall genes encoding ribosomal protein genes during the indicated stages.X axis shows the relative expression of these genes in Low Protein IVFembryos, compared to Control. Grey line shows distribution of dietaryeffects on all non-RPG genes, for all four stages. Left shift at the2-cell stage shows downregulation of RPGs in Low Protein 2-cell embryos.FIGS. 12B-E show GSEA plots for various sets of genes involved inribosome biogenesis at the indicated developmental stages. FIG. 12Fshows an example image of a blastocyst stained with DAPI and anti-Cdx2to image total cell number and trophectoderm cells. FIG. 12G shows thatLow Protein diet reproducibly alters developmental tempo. FIG. 12H showsaggregated data for three replicate experiments, showing the number ofblastocysts with the indicated number of cells, for embryos generatedvia IVF using Control or Low Protein sperm, as indicated.

FIGS. 13A-13H show dietary effects on tRNAs in testes. FIG. 13A shows aschematic illustrating assay for tRNA charging analysis. FIG. 13B showsvalidation of tRNA charging protocol. Changes in tRNA abundance forcharged and uncharged tRNAs are shown on the y axis, sorted by thechange in charged tRNA abundance. FIG. 13C shows testicular tRNAabundance correlation with codon bias in the mouse. The X axis showsintact tRNA abundance in testis in log scale, and the y axis shows thecorresponding codon abundance (in codon frequency/1000) in all murinemRNAs, or in the 47 most-highly expressed mRNAs in testis. FIG. 13Dshows validation of tRNA charging analysis. Scatterplot shows abundanceof approximately 60-80 nt RNAs in the total RNA protocol (x axis, logscale) compared to abundance of RNAs in the charged tRNA protocol (yaxis, log scale). FIGS. 13E-13G show Low Protein vs. Control effects ontRNA levels for total (FIG. 13E), uncharged (FIG. 13F), and charged(FIG. 13G) tRNA levels in testis. FIG. 13H shows that dietary effects onsperm tRFs are not explained by effects on intact tRNA abundance intestes.

FIGS. 14A and 14B show that there are consistent dietary effectsthroughout the reproductive tract. FIG. 14A shows the dietary effects onsmall RNA abundance in testes and caput and cauda epididymis samples.Each heatmap shows log 2 of Low Protein/Control RNA abundance for a pairof samples, showing RNAs (rows) that exhibit consistent dietary effectsacross >75% of samples. FIG. 14B shows the coherent dietary effects ontRF-Gly and let-7 family members throughout the male reproductive tract.For each RNA, bars show average and standard error of the mean for LowProtein effects on the abundance of the RNA species in the indicatedtissue. Changes with a nominal p value of <0.05 (paired t test, notcorrected for multiple testing) are indicated with asterisks.

FIGS. 15A-15F show RNA populations in caput sperm. FIG. 15A shows thatunwashed caput sperm are contaminated with RNAs abundant in caputepididymosomes. FIG. 15B shows a comparison of small RNA payloads ofcauda vs. caput sperm for all RNA species with an abundance of at least1 ppm in both sperm populations. FIG. 15C shows the proximal-distalbiases observed for epididymis (x axis) are recapitulated in cauda vs.caput sperm samples (y axis). FIG. 15D shows that there is a gain in allfour tRFs from caput to cauda. Data from FIG. 15D are shown withtRF-Val-CAC normalized to tRF-Glu-CTC rather than to microRNAs. FIG. 15Eshows that tRF-Val-CAC is strongly cauda-enriched in all threepreparations—epididymal epithelium, epididymosomes, and sperm—examined.FIG. 15F depicts Northern blots showing that caput sperm carry intacttRNAs

FIGS. 16A-16C show that dietary information is carried in sperm. FIG.16A shows the sperm from males consuming Control or Low Protein dietwhich were used to fertilize oocytes gathered from Control females. Sqlelevels (normalized to Actb) are shown for all offspring as individualpoints, with horizontal lines showing mean expression. FIG. 16B showsthe cumulative distribution of Sqle expression for all offspringgenerated using Control or Low Protein sperm, as indicated. FIG. 16Cshows consistent litter effects. Sqle levels were averaged for alloffspring of a given litter.

FIGS. 17A and 17B show the mechanistic basis for tRF-Gly-GCC regulationof MERVL. FIG. 17A shows RNA-Seq and ribosome footprinting data forSp110. FIG. 17B shows that RNA abundance and ribosome footprinting dataare highly correlated, indicating that tRF-Gly-GCC does not affect MERVLelements as a secondary effect of its effects on protein translation.

FIGS. 18A-18D show the reconstitution of small RNA delivery totesticular sperm. FIG. 18A depicts an experimental schematic showingpurified testicular sperm, which carry extremely low levels of tRFs,were incubated with caput epididymosomes for 2 hours, and thenextensively washed with detergent. Small RNAs were purified from eithermock-treated testicular sperm, or reconstituted sperm, and deepsequenced. Pie charts show average levels of various small RNA classes,revealing increased levels of microRNAs and tRFs delivered to testicularsperm by epididymosomes. FIG. 18B shows the delivery of two prominenttRFs to testicular sperm. Taqman q-RT-PCR for the indicated tRFs, withindividual replicates plotted (on aa log 2 y axis) relative to theaverage level present in Mock-treated testicular sperm. FIG. 18C showsthe distribution of small RNA changes upon reconstitution. The X axisshows the log 2-fold difference between reconstituted and mock-treatedsperm; positive values indicate delivery by epididymosomes. The peak forpiRNAs is approximately −0.6, reflecting the assumption in genome-widenormalization of equal numbers of molecules in both samples. Underconditions of RNA delivery, pre-existing RNAs (shown), the piRNAs thatpredominate in testicular sperm will appear to “decrease” in abundancedue to normalization. As such, all values over −0.6 indicate gain of RNAduring the fusion protocol.

FIG. 18D shows a scatterplot of small RNA abundances from deepsequencing data. The strong diagonal for piRNAs indicates RNAs presentin testicular sperm that are not affected by the delivery process.Essentially all RNAs shown either lie along this diagonal, indicatingthat they are either absent or present in low abundance in caputepididymosomes, or above the diagonal, indicating widespread delivery ofmany RNA species to testicular sperm during reconstitution.

DETAILED DESCRIPTION

The present disclosure is directed to methods and compositions that canalter sperm molecular content, such that a disease, disorder, orcondition that would otherwise be inherited by offspring, are not, orsuch disease, disorder, or condition severity is reduced.

The inventors discovered that as sperm mature in the male reproductivetract, their molecular cargo changes. Specifically, as sperm movethrough the epididymis, the small RNA molecule (sRNA) content changesdramatically. The sperm sRNA content mirrors that found in vesiclesfound in the different regions of the epididymis (“epididymosomes”). Forexample, a sperm located in the cauda epididymis has a similar sRNAcontent as a caudal epididymosome.

The inventors have discovered methods of altering the RNA content ofsperm, such as to increase the maturity of immature sperm, to “rescue”defective mature sperm that have at least one sRNA defect (e.g., areduction or absence of at least one sRNA, or at least one aberrantsRNA), and to decrease transmission of epigenetically-transmitteddiseases or disorders to progeny. These methods involve contacting thetarget sperm with vesicles, such as epididymosomes, to alter the spermRNA content. Such treated sperm can then be used to fertilize oocytes invitro or in vivo.

I. Definitions

As used in the specification and in the claims, the term “comprising”may include the embodiments “consisting of” and “consisting essentiallyof” The terms “comprise(s),” “include(s),” “having,” “has,” “may,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that require thepresence of the named ingredients/steps and permit the presence of otheringredients/steps. However, such description should be construed as alsodescribing compositions or processes as “consisting of” and “consistingessentially of” the enumerated compounds, which allows the presence ofonly the named compounds, along with any pharmaceutically acceptablecarriers, and excludes other compounds.

As used herein, approximating language may be applied to modify anyquantitative representation that may vary without resulting in a changein the basic function to which it is related. Accordingly, a valuemodified by a term or terms, such as “about” and “substantially,” maynot be limited to the precise value specified, in some cases. In atleast some instances, the approximating language may correspond to theprecision of an instrument for measuring the value. The modifier “about”should also be considered as disclosing the range defined by theabsolute values of the two endpoints. For example, the expression “fromabout 600 to about 2000” also discloses the range “from 600 to 2000.”The term “about” may refer to plus or minus 10% of the indicated number.For example, “about 10%” may indicate a range of 9% to 110%, and “about1” may mean from 0.9 to 1.1. Other meanings of “about” may be apparentfrom the context, such as rounding off, so, for example “about 1” mayalso mean from 0.5 to 1.4.

As used herein, “treatment” or “treating,” is defined as the applicationor administration of a therapeutic agent to a subject, or application oradministration of a therapeutic agent to an isolated tissue, cell line,or cell from a subject. “Therapeutic agents” include vesicles, includingepididymosomes.

As used herein, “patient,” “individual” or “subject” refers to a humanor a non-human mammal. Non-human mammals include, for example, livestockand pets, such as ovine, bovine, porcine, canine, feline and murinemammals. Non-human mammals also include primates. Preferably, thepatient, subject, or individual is human.

As used herein, “untreated sperm” means sperm that have not beensubjected to the application or administration of a therapeutic agent asdescribed in the disclosed methods. “Treated sperm” means sperm thathave been subjected to the application or administration of atherapeutic agent as described in the disclosed methods. In embodiments,an untreated sperm may have been exposed to vesicles in the malereproductive tract, but become treated sperm when exposed to vesiclesthat are autologous or heterologous in vitro. In some embodiments, theuntreated sperm are treated in vivo when exposed to autologous orheterologous vesicles, which can be comprised in a composition, such asa pharmaceutical composition.

As used herein, “vesicle” means extracellular vesicles (EVs) that cellsshed from their plasma membrane, or from multivesicular bodies. Thesevesicles are generally referred to as microvesicles, ectosomes, sheddingvesicles, or microparticles, as well as exosome vesicles (or exosomes).Exosomes are extracellular vesicles that originate from multivesicularendosomes (MVEs) that fuse with the plasma membrane. However,circulating extracellular vesicles such as epididymosomes also includemicrovesicles (MVs). Thus, unless otherwise noted, the term “vesicles”includes MVs and exosomes. A vesicle comprises at least one RNAmolecule, such as a small RNA (sRNA). Vesicles that originate fromspecific tissues or cells can be designated by specific terms, such asepididymosomes, which originate from the epididymis; seminosomes whichoriginate from seminal fluid, and prostasomes, which originate from theprostate. The ExoCarta database (found on the world-wide web atexocarta.org) contains the proteins, lipids, and RNA that have beenfound in EVs from various sources.

As used herein, “altering the RNA content” means, such as when appliedto cells, such as sperm, to add or remove an RNA molecule by treatingthe cells or sperm. For example, vesicles can be used to deliver RNAcargo to sperm, thus altering the RNA content of the sperm.

As used herein, “increasing sperm maturity” means that after atreatment, the sperm takes on or improves in at least one characteristicthat indicates that the sperm has further matured. An increase in spermmaturity is reflective of a healthy sperm that has progressed to thesame location or further in the male reproductive tract relative to theuntreated sperm.

As used herein, “a defective sperm” means a sperm that lacks at leastone characteristic in relation to its maturity by virtue of its locationin the male reproductive tract or ejaculate when compared to a healthyejaculated sperm. The altered characteristic can be a difference in atleast one molecule, such as an RNA molecule or a polypeptide. Thedifference can be the absence of a molecule, the presence of a moleculethat is usually absent in healthy sperm, or a changed molecule, such asa mutated or mis-processed molecule. In some embodiments, the at leastone molecule is a sRNA. “Rescuing a defective sperm” means to add orsubtract the molecule that is different than healthy sperm, or supplyinga wild-type molecule of a changed molecule, to the sperm by treating thesperm, so that the sperm resemble healthy sperm in relation to itssource of isolation from the male reproductive tract or ejaculate.“Defective mature sperm” means a sperm that appears to have matured byvirtue of it completing its journey through the male reproductive tract,but lacks at least one characteristic of mature healthy sperm. Adefective mature sperm is not necessarily incapable of fertilizing anoocyte, but may instead transmit a trait, condition, disease, ordisorder to a resulting progeny.

As used herein, “epigenetically-transmitted” means a trait, condition,disease, or disorder transmitted by a parent to offspring wherein theacquired trait, condition, disease, or disorder is not the result of amutation in DNA; that is, the trait is transmitted in violation ofMendelian genetics. In such intergenerational epigenetic inheritance,epigenetic phenotypes are transmitted to at least one generation and maybe gender-specific.

As used herein, “healthy sperm” means a sperm that has thecharacteristics of sperm found in healthy, fertile subjects in relationto its maturity by virtue of its location in the male reproductive tractor ejaculate.

As used herein, “sRNA” means “small RNA” and includes all classes ofsmall RNAs, including: small interfering RNAs (siRNAs), Piwi-interactingRNAs (piRNAs), microRNAs (miRNAs), tRNA fragments (tRF), small nucleolarRNA (snoRNA), small rDNA-derived RNA (srRNA), and small nuclear RNA(U-RNA). Generally, sRNAs are about 200 nucleotides or less in length,such as 40 nucleotides in length, or less. siRNAs are generallydouble-stranded pairs of RNAs about 20-25 base pairs long, and canparticipate in the RNA interference (RNAi) pathway (Hannon, G J and J JRossi. 2004. Nature, 431:371-378). piRNAs are non-coding RNA moleculesof about 26-31 nucleotides long and form RNA-polypeptide complexes withpiwi proteins. These RNA molecules have been linked to epigenetic genesilencing of “molecular parasites,” such as transposons found in germline cells (Czech, B and G J Hannon. 2016. Trends Biochem Sci, 41:324-337; Siomi M C, et al. 2011. Nat Rev Mol Cell Biol, 12:246-258).miRNAs are about 19-24 nucleotide long, non-coding RNA molecules. Theyregulate protein-coding gene expression translationally andpost-transcriptionally (Virant-Klun, I., et al. 2016. Stem Cells Int.2016:3984937). tRFs are fragments of tRNA molecules that are about 28 to34 nucleotides long, have a wide variety of molecular effects on cellsand are found enriched in, for example, sperm (Peng, H., et al. 2012.Cell Res, 22: 1609-1612). snoRNA guide chemical modifications of otherRNAs, such as rRNAs, tRNAs, and snRNAs; these small non-coding RNAs fallinto two classes, one of about 60-90 nucleotides long (“box C/D”), andanother of about 120-140 nucleotides long (“box H/ACA”)(Dupuis-Sandoval, F, et al. 2015. Wiley Interdiscip Rev RNA, 6:381-397). srRNAs map by sequence to rRNA coding regions in the sensedirection; coimmunoprecipitate with Argonaute proteins, and are involvedin various signaling pathways, and are thought to be about 18-30nucleotides long (Wei, H, et al. 2013. PLoS One, 8: e56842). U-RNAmolecules are about 150 nucleotides long and function to processpre-mRNA in the nucleus (Zhang, L, et al. 2013. Protein Sci, 22:677-692).

An “aberrant” sRNA is an sRNA molecule that differs from a wild-typesRNA. For example, the sRNA has a changed sequence, such as one or morepoint mutations, deletions, insertions, translocations; or is chemicallymodified, etc.

An “artificial RNA” or “synthetic RNA” is an RNA molecule that issynthesized in vitro by any art-accepted method.

As used herein, “stress” means a state of physical, mental or emotionalstrain or tension in an organism, such as a subject, that results fromadverse or demanding circumstances and causes physiological alterationsin the organism. The stress is often applied repeatedly or continually.In some embodiments, the stress may last for a period of time. In somecases, the physiological alterations are present after the stress hasbeen applied.

As used herein, “dietary restriction” means a diet that is deficient inone or more components of a healthy diet, such as a vitamin, a nutrient,a micronutrient, a fat, a simple carbohydrate, a complex carbohydrate,and protein, or a calorie deficit (that is, insufficient calories tosupport the health of an organism) such that the physiology of anorganism is altered. In some embodiments, the restriction is repetitiveor continual. In some embodiments, the restriction may last for a periodof time. In some cases, the physiological change persists after thedietary restriction stops.

As used herein, “overeating” means the condition of an organismconsuming more calories than is necessary to maintain the normal healthof the organism.

As used herein, “stress-related disease or disorder” means a disease ordisorder which symptoms in an organism can be triggered or amplified bythe application of stress to an organism. In some embodiments, astress-related disease or disorder is related to mental health. In someembodiments, the mental health disease or disorder is a form ofdepression, such as major depressive disorder (also known as majordepression or clinical depression), dysthymia, and bipolar disorder(having a depressive phase). Other examples of mental health disease ordisorders include those that are anxiety-based conditions, includinggeneralized anxiety disorder, a specific phobia, social anxietydisorder, separation anxiety disorder, agoraphobia, and panic disorder.

As used herein, “metabolic disorder” means a disorder wherein acomponent of metabolism is absent, up-regulated, or down-regulated whencompared to the metabolism of a healthy organism, such as a subject. Ametabolic disorder can manifest in many forms. For example, themetabolic disorder can be a hepatic metabolic disorder, which originatesin the liver, or affects the expression of a marker of liver-basedmetabolism, such as Sqle gene expression. A manifestation of a hepaticmetabolic disorder includes a reduction in sterol biosynthesis, such asreduced cholesterol biosynthesis. Pancreatic metabolic disorders includetype II diabetes.

As used herein, “reduced” means that the substance or activity beingmeasured is present in a lesser amount and/or lesser activity than whencompared to that of a healthy organism.

“Healthy” means that the organism, tissue, or cell has the compositionand activity of an organism, tissue or cell that falls within theboundaries of wild-type expression, indicative of a non-disease state.

II. Methods

In an aspect, disclosed herein is a method of altering the RNA contentof a sperm of a subject, comprising contacting the sperm with a vesiclecomprising a sRNA to produce a sperm having an altered RNA content.

In a second aspect, disclosed herein is a method of treating anepigenetically inheritable trait at risk of being transmitted to aprogeny of a subject, comprising altering the RNA content of a spermfrom the subject by contacting the sperm with a vesicle comprising asRNA and fertilizing an oocyte with the sperm to produce the progeny.

In embodiments, the sRNA is selected from the group consisting of asiRNA, a miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNAfragment. In further embodiments, the tRNA fragment is selected from thegroup consisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, atRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment,a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In an embodiment,the vesicle is an exosome; in yet further embodiments, the exosome is anepididymosome; the epididymosome can be selected from the groupconsisting of caput epididymosome, corpus epididymosome, and caudaepididymosome. In other embodiments, the vesicle is a seminosome or aprostasome. In other embodiments, the vesicle is a microvesicle.

In some embodiments, prior to contacting the sperm, the sperm isimmature and altering the RNA content increases sperm maturity. In otherembodiments, prior to contacting the sperm, the sperm is defective andaltering the RNA content diminishes at least one defect. In suchembodiments, the defective sperm can comprise a defect selected from thegroup consisting of a reduced level of sRNA, at least one aberrant sRNA,or absence of at least one sRNA that is present in healthy mature sperm.In an embodiment, the defective sperm comprises a defect in siRNA,miRNA, piRNA, snoRNA, srRNA, U-RNA, and tRNA fragment content. Infurther embodiments, the tRNA fragment content comprises a defectselected from the group consisting of tRNA-Gly-CCC fragment content,tRNA-Gly-TCC fragment content, tRNA-Gly-GCC fragment content,tRNA-Val-CAC fragment content, tRNA-Glu-CTC fragment content,tRNA-Lys-CTT fragment content, and tRNA-His-GTG fragment content. Inother embodiments, the defective sperm comprises a decrease in at leastone let-7 species of RNA when compared to a healthy sperm.

In embodiments, after altering the RNA content, the sperm fertilizes anoocyte.

In an embodiment, the subject is a mammal, such as a primate, such as ahuman.

In embodiments, the sperm that is altered is obtained from the subject'scaput epididymis, corpus epididymis, cauda epididymis, vas deferens,testis, or ejaculate. In further embodiments, the sperm is obtained fromthe subject's caput epididymis, corpus epididymis, or cauda epididymisusing microscopic or microsurgical epididymal sperm aspiration (MESA) orpercutaneous epididymal sperm aspiration (PESA). In yet other furtherembodiments, the sperm is obtained from the subject's testis using atechnique selected from the group consisting of needle aspiration(TESA), percutaneous or open surgical biopsy (TESE), multibiopsy TESE,microdissection TESE, site-directed TESE after fine needle aspirationmapping, and MicroTESE. Such techniques are routinely used in assistedreproduction.

In embodiments, the subject which sperm is altered is experiencing acondition selected from the group consisting of a stress-related diseaseor disorder, dietary restriction, and obesity. In an embodiment, thedietary restriction is protein deficiency. In another embodiment, thestress-related disease or disorder is selected from the group consistingof major depressive disorder, dysthymia, bipolar disorder, generalizedanxiety disorder, a phobia, social anxiety disorder, separation anxietydisorder, agoraphobia, and panic disorder.

In embodiments, the vesicle that is contacted to the sperm isheterologous to the subject. In other embodiments, the vesicle isautologous to the subject. In yet other embodiments, the vesiclecomprises a heterologous RNA; the heterologous RNA can comprise a smallRNA (sRNA). Such sRNA can be one selected from the group consisting of asiRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNA fragment.In embodiments where the sRNA is a tRNA fragment, the tRNA fragment canbe selected from the group consisting of a tRNA-Gly-CCC fragment, atRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment,a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTGfragment. In other embodiments, the vesicle comprises autologous RNA.Such an vesicle can comprise sRNA that can be selected from the groupconsisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and atRNA fragment. In the case where the sRNA is a tRNA fragment, the tRNAfragment can be selected from the group consisting of a tRNA-Gly-CCCfragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, atRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment,and a tRNA-His-GTG fragment. In other embodiments, the vesicle comprisesan artificial (synthetic) RNA. In such vesicles, the sRNA can beselected from the group consisting of a siRNA, miRNA, a piRNA, a snoRNA,a srRNA, a U-RNA, and a tRNA fragment. In the case where the sRNA is atRNA fragment, then the tRNA fragment can be selected from the groupconsisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, atRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment,a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In otherembodiments, the vesicle comprises a transgene.

In embodiments, the altered sperm fertilizes an oocyte in vitro. Inother embodiments, the sperm is used in intracytoplasmic sperm injection(ICSI). These embodiments can further comprise implanting the fertilizedoocyte to a second subject to produce a progeny.

In other embodiments, the altered sperm fertilizes an oocyte in vivo.

In embodiments, prior to contacting the sperm with a vesicle, the spermare frozen.

Specifically in the second aspect, the epigenetically inheritable traitis a disease or disorder that is a metabolic or stress-related diseaseor disorder. In some embodiments, the metabolic disease or disordercomprises a glucose or hepatic metabolic disease or disorder. In furtherembodiments, the hepatic metabolic disease or disorder comprises reducedsterol biosynthesis. In yet further embodiments, the reduced sterolbiosynthesis comprises reduced cholesterol biosynthesis. In even furtherembodiments, hepatic Sqle gene expression is upregulated. In otherembodiments, the stress-related disease or disorder is selected from thegroup consisting of major depressive disorder, dysthymia, bipolardisorder, generalized anxiety disorder, a phobia, social anxietydisorder, separation anxiety disorder, agoraphobia, and panic disorder.

In embodiments of this second aspect, the progeny lacks symptoms of theepigenetically inheritable trait. In other embodiments, the progeny hasameliorated symptoms of the epigenetically inheritable trait.

In yet other embodiments of this second aspect, the subject isexperiencing a condition selected from the group consisting of astress-related disease or disorder, dietary restriction, and obesity. Infurther embodiments, the dietary restriction is protein deficiency. Inother further embodiments, the stress-related disease or disorder isselected from the group consisting of major depressive disorder,dysthymia, bipolar disorder, generalized anxiety disorder, a phobia,social anxiety disorder, separation anxiety disorder, agoraphobia, andpanic disorder.

Vesicles

Vesicles can be used to treat a subject, organ, tissue, or cell (such assperm). Vesicles can be used as found in their normal milieu, such as atissue fluid. In the case of epididymosomes, fluid found in theepididymis contains vesicles and can be directly applied to the targetsubject, organ, tissue, or cell (such as sperm).

However, in preferable embodiments, vesicles are used at least partiallypurified. “Purified” means to be substantially free from othercomponents normally associated with the purification target in a nativeenvironment. Vesicle purification can be accomplished by manyprocedures. For example, in the case of cultured cells, differentialultracentrifugation can be used (Raposo, G and W Stoorvogel. 2013. JCell Biol., 200: 373-383). If cultured cells are used as a source ofvesicles, media components, such as serum (e.g., fetal bovine serum),are depleted of EVs before applying to the cells so as to notcontaminate the cell vesicle preparation with vesicles of other origins.To separate vesicles from non-membranous particles (such as proteinaggregates), the relatively low buoyant density and differences infloatation velocity can be used (Raposo, G, et al. 1996. J Exp Med.,183: 1161-1172; Escola, J M, et al. 1998. J Biol Chem, 273: 20121-20127;Van Niel, G, et al. 2003. Gut, 52:1690-1697; Wubbolts, R, et al. 2003. JBiol Chem, 278: 10963-10972; Aalberts, M, et al. 2012. Biol Reprod,86:82. In some embodiments, vesicles can be further purified throughimmunopurification by using a protein of interest found on the surfaceof the target vesicle. A method of purifying vesicles is also set out inthe Examples.

Vesicles can also be isolated using a number of commercially availablekits, such as Total Exosome Isolation (Invitrogen (ThermoFisherScientific); Waltham, Mass.), ExoQuick-TC™ (System Biosciences; PaloAlto, Calif.), ME™ (New England Peptide; Gardner, Mass.), miRCURY™(Exiqon; Woburn, Mass.); and Exo-spin™ (Cell Guidance Systems; St.Louis, Mo.),

In some embodiments, physical methods can be used to producenanovesicles that have some of the features of vesicles (Gyorgy, B. etal. 2015. Annu Rev Pharmacol Toxicol. 55: 439-464). For example, cellscomprising the target molecules to be transferred to the target subject,organ, tissue, or cell can be extruded through filters, which fragmentthe cells and generate vesicles (Jang, S C, et al. 2013. ACS Nano, 7:7698-7710). Alternatively, such cells can be extruded through amicrofluidic chamber (Jo, W, et al. 2014. Lab Chip, 14: 1261-1269). Suchformed vesicles are similar to endogenous EVs in size, shape, andcomposition and can deliver RNA molecules (Gyorgy, B. et al. 2015. AnnuRev Pharmacol Toxicol. 55: 439-464). In principle, vesicles can also beformed by sonicating, lysing, electroporating and freeze-thawing cells(Gyorgy, B. et al. 2015. Annu Rev Pharmacol Toxicol. 55: 439-464).

In certain embodiments, vesicles (e.g., epididymosomes) of the inventionare between about 50 nm and about 400 nm in diameter, between about 60nm and about 350 nm in diameter, between about 70 nm and about 300 nm indiameter, between about 80 nm and about 250 nm in diameter, betweenabout 90 nm and about 150 nm in diameter, between about 110 nm and about180 nm in diameter, between about 240 nm and 360 nm in diameter, or anyranges of diameters or individual diameters between these ranges. Inexemplary embodiments, vesicles (e.g., epididymosomes) of the inventionare approximately 150 nm in diameter.

In embodiments, vesicles can be loaded with molecular cargo (such as asRNA), such as by using electroporation or co-incubation(Alverez-Erviti, L, et al. 2011. Nat. Biotechnol. 29: 341-345;El-Andaloussi, S, et al. 2012. Nat Protoc, 7: 2112-2126; Sun, D, et al.2010. Mol. Ther. 18:1606-1614; Tian, Y H, et al. 2014. Biomaterials,35:2383-2390). In some embodiments, the molecular cargo includes atransgene. In other embodiments, the molecular cargo includes anartificial (synthetic RNA). In some embodiments, the molecular cargoincludes an autologous molecule, such as an RNA molecule (such as asRNA); in other embodiments, the molecular cargo includes a heterologousmolecule.

In some embodiments, vesicles can be modified to incorporate a moleculethat targets a tissue or cell-specific molecule; e.g., a molecule thatbinds to a sperm-specific membrane molecule, such as a protein or alipid.

In some embodiments, further characterization of the vesicles may bedesired to ensure that the targeted vesicles have indeed been purified.Methods such as sizing (although both “true” vesicles as well as MVs canbe isolated in a single preparation; size does not necessarilydistinguish true vesicles from MVs), immunoblotting, mass spectrometry,and imaging techniques can be used to further characterize isolatedvesicles (Raposo, G and W Stoorvogel. 2013. J Cell Biol., 200: 373-383).Imaging techniques include conventional transmission electronmicroscopy, whole mount transmission electron microscopy, andcryo-electron transmission electron microscopy. In addition,nanoparticle tracing analysis to determine size distribution of theisolated vesicles can be accomplished based on the Brownian motion ofvesicles in suspension (Soo, C Y, et al. 2012. Immunology, 136:192-197). Furthermore, individual vesicles can be analyzed using highresolution flow cytometry methods when the vesicles are immunolabeled(Nolte-'t Hoen, E N. 2012. Nanomedicine, 8:712-720; van der Vlist, E J,et al. 2012. Nat Protoc, 7: 1311-1126).

Sperm Acquisition

In embodiments, untreated sperm, or untreated immature sperm, oruntreated defective mature sperm (“sperm”) are obtained from a subject,such as from a mammal, such as a primate or human. A mammalian sperm,which may also be referred to as a “spermatid,” “spermatozoon” or“spermatozoan,” are produced through spermatogenesis inside the testiclethrough meiotic division. Sperm formed in the testis then enter thecaput epididymis, progress through the corpus epididymis region, andfinally enter the cauda epididymis. After exiting the testis, spermmature by structurally and functionally reorganizing the sperm membrane,which maturation results in the acquisition of motility andfertilization capabilities. However, sperm also lose their ability tosynthesize proteins (Barkalina, N, et al. 2015. Human Reprod Update,21(5): 627-639). Epididymosomes fuse with sperm to deliver proteins,including P34H (necessary for fertilization), ADAM-7 (a disintegrin andmetalloproteinase), glioma pathogenesis-related I-like protein;epididymal sperm binding protein I (ELSPBPI), and plasma membraneCa²⁺-ATPase (Barkalina, N, et al. 2015. Human Reprod Update, 21(5):627-639). Fusion not only changes the protein composition of the sperm,but also its lipid composition (Barkalina, N, et al. 2015. Human ReprodUpdate, 21(5): 627-639).

During ejaculation, sperm flow from the cauda epididymis through the vasdeferens prior to entering the ejaculatory duct. The sperm then passthrough the prostate gland, enter the urethra, and exit the body throughthe urethral opening in the seminal fluid (also referred to as theejaculate). Sperm for use in the disclosed methods can be retrieved fromany point along the reproductive tract from the testis to the ejaculate,including the subject's testis, epididymis (including the caput, corpus,or cauda epididymis), vas deferens, or ejaculate. In the case of in vivofertilization, the sperm remain in the ejaculate for fertilization ofthe oocyte.

In one embodiment, sperm can be obtained from a subject's epididymis(including from the caput, corpus, and cauda epididymis) usingmicroscopic or microsurgical epididymal sperm aspiration (MESA) orpercutaneous epididymal sperm aspiration (PESA). In another embodiment,sperm can be obtained from a subject's testis using a technique selectedfrom the group consisting of needle aspiration (TESA), percutaneous oropen surgical biopsy (TESE), multibiopsy TESE, microdissection TESE,site-directed TESE after fine needle aspiration mapping, and MicroTESE.Such techniques are routinely used in assisted reproduction.

In one embodiment, sperm can be from the same subject or a differentsubject than the source of vesicles for use in the disclosed methods. Inanother embodiment, the sperm can be a donor sperm, such as thoseavailable from a sperm bank.

The isolated, untreated sperm may comprise a condition such as reducedlevels of sRNA, at least one aberrant sRNA, or the absence of at leastone sRNA that is present in healthy sperm. In other embodiments, theremay be increased levels of an sRNA compared to healthy sperm, or thepresence of sRNAs that are not usually present in healthy sperm. Forexample, untreated sperm can have an absence or decrease in at least onesRNA selected from the group consisting of tRNA-Gly-CCC fragments,tRNA-Gly-TCC fragments, tRNA-Gly-GCC fragments, tRNA-Lys-CTT fragments,and tRNA-His-GTG fragments; or aberrant forms of these molecules. Adecrease in a sRNA is one wherein such a decrease has an effect on anoffspring, such as in the case of an epigenetically transmittedcondition. In some embodiments, the sRNA that is absent, decreased, oris aberrant is a let-7 species of miRNA.

In some embodiments, the sperm that is obtained is from a subject thathas experienced some form of stress, including mental stress, dietaryrestriction, or overeating. For example, the subject may suffer from aprotein deficiency. In other embodiments, the subject has a disease ordisorder that is a metabolic or stress-related disease or disorder. Suchdiseases and disorders can be a hepatic metabolic disease or disorder,which can include reduced sterol biosynthesis (such as reducedcholesterol synthesis), or an upregulation or downregulation in hepaticSqle gene expression. In other embodiments, the stress-related diseaseor disorder is a mental health disease or disorder, such as depression.

In some embodiments, the sperm is frozen using well-establishedtechniques, such as those used by sperm banks, for later use in thedisclosed methods.

Oocyte Fertilization

A mammalian “oocyte,” which may also be referred to as an “ovocyte,”“immature ovum” or “egg cell” for use in the disclosed methods isproduced through oogenesis by meiotic division.

An oocyte for use in in vitro fertilization can be retrieved from asubject by any known method, including aspiration directly from theovarian follicles. An oocyte for use in in vivo fertilization is notretrieved, but fuses with the sperm within the subject prior toimplantation in the uterus.

An oocyte may be fertilized by any known method, including in vivomethods and in vitro methods. In certain embodiments, the method ofoocyte fertilization may be in vitro fertilization (IVF). In oneembodiment, the oocyte may be fertilized through intracytoplasmic sperminjection (ICSI).

Vesicle Application

For in vitro application, partially or fully purified vesicles can beresuspended in a buffered solution, such as phosphate-buffered saline(PBS) or cell culture media (and if serum is present, it isEV-depleted). Alternatively, the vesicles can be formulated into apharmaceutical composition comprising, for example, a pharmaceuticalexcipient or carrier. To contact the target tissue or cells, forexample, the vesicles are applied to the tissue or cells, and thecomponents incubated for a sufficient time to permit vesicle fusion andcargo delivery. For example, solutions comprising vesicles can beincubated with the target tissue or cells for minutes to hours to days,such as, in minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 120, 180, and 240; such as in hours, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, 42,and 48; such as in days, 3 and 4. One of skill in the art can ascertainthe time to incubate an vesicle-containing composition with the targettissue or cells, adjusting variables of not only time, but alsovariables concerning the applied concentration of vesicles (oftendetermined by quantifying total protein), temperature (typicallyphysiological temperatures, but above about 4° C.), volume, componentsof buffered solution used for resuspension, and number of target cells.Vesicles can be applied to the target tissue or cells multiple times.

Alternatively, vesicles can be injected into a subject in apharmaceutical composition, such as glucose (e.g., 5% glucose) (Cooper,J M, et al. 2014. Mov Disord., 29(12): 1476-1485).

Pharmaceutical compositions comprising vesicles, dosage forms, anddosing, are described in more detail below.

As defined above, vesicles comprise at least one RNA molecule. Such RNAmolecule can be a sRNA, such as siRNA, miRNA, piRNA, snoRNA, srRNA,U-RNA, or tRNA fragment (tRF). Such RNA can be heterologous orautologous. For example, in some embodiments, the sperm to be treatedare immature, whether by virtue of a biological cause or isolationlocation, but the vesicles are derived from cauda epididymis of the samesubject (e.g., wherein the RNA are therefore autologous in the case ofisolating immature sperm based on location), or derived from the caudaepididymis of another subject (thus comprising heterologous RNA), whichmay be desirable to treat immature sperm from healthy subjects as wellas for treating immature sperm from subjects having a biological causethat results in immature sperm. In other embodiments, heterologous RNAis introduced into vesicles isolated from the same subject as the spermdonor. For example, such RNA can be one that is derived from theepididymis, such as the cauda epididymis.

Pharmaceutical Compositions

Pharmaceutical compositions comprising vesicles are expounded on inpart, for example, in US 20160060652.

Pharmaceutical compositions that contain vesicles useful in thedisclosed methods can comprise a liquid medium. Examples of liquid mediainclude water, physiologically acceptable buffer solutions(phosphate-buffered saline, etc.) and biocompatible aqueous mediums suchas propylene glycol and polyoxyethylene sorbitan fatty acid ester. Themedia is desirably sterile and adjusted to be isotonic to blood or othertissue fluid (e.g., epididymal) if necessary.

Pharmaceutical compositions can comprise a pharmaceutically acceptablecarrier. Examples of pharmaceutically acceptable carriers includesuspending agents, tonicity agents, buffers and preservatives. Carrierscan be used to facilitate formulation and maintaining the dosage formand drug effects.

For example, glyceryl monostearate, aluminum monostearate,methylcellulose, carboxymethylcellulose, hydroxymethylcellulose andsodium lauryl sulfate can be used as suspending agents. Examples oftonicity agents include sodium chloride, glycerin and D-mannitol.Examples of buffers include phosphate, acetate, carbonate and citrate.Examples of preservatives include benzalkonium chloride,parahydroxybenzoic acid and chlorobutanol.

If necessary or desired, pharmaceutical compositions can also comprise acorrigent, a thickener, a solubilizing agent, a pH adjuster, a diluent,a surfactant, an expander, a stabilizer, an absorption promoter, awetting agent, a humectant, an adsorbent, a coating agent, a colorant,an antioxidant, a flavoring agent, a sweetener, an excipient, a binder,a disintegrant, a disintegration inhibitor, a filler, an emulsifier, aflow control additive, or a lubricant.

Pharmaceutical compositions useful in the disclosed methods can alsocontain an additional drug without losing pharmacological effectspossessed by the vesicles. For example, the pharmaceutical compositionmay contain an antibiotic.

Information directed to suitable formulations and additional carrierscan be found in, for example, Remington “The Science and Practice ofPharmacy” (20th Ed., Lippincott Williams & Wilkins, Baltimore Md.),which is incorporated by reference in its entirety herein.

In some embodiments, a dosage form may be desired. A dosage form of thepharmaceutical composition is not limited and can be any form that doesnot inactivate the vesicle or its contents. The dosage form ofpharmaceutical compositions can be, for example, a liquid, solid orsemisolid form. Specific examples of dosage form include parenteraldosage forms such as injections, suspensions, emulsions, creams,ointments, gels and foams. In some embodiments, the dosage form is avaginal gel or foam.

A “pharmaceutically effective amount” refers to a dose required for thevesicles contained in pharmaceutical compositions to prevent, diminish,or treat the target disease or condition, or alleviate symptoms, in asubject and/or in the subject's offspring (or in some cases, theoffspring's offspring). A specific dose differs depending on the diseaseto be prevented, diminished, and/or treated; the mechanism of actionunderlying the occurrence of the disease, the dosage form used,information about a subject and an administration route, etc. The rangeof the pharmaceutically effective amount and a preferred administrationroute of the pharmaceutical composition that is administered to a humansubject are generally set on the basis of data obtained from cellculture assay and animal experiments. The final dose can be determinedand adjusted by the judgment of, for example, a physician. Informationabout the subject to be taken into consideration can include the degreeof progression or severity of the disease, general health conditions,age, body weight, sex, diet, drug sensitivity and resistance totreatment, etc.

The pharmaceutical compositions can be administered twice or more atpredetermined intervals of time, for example, every hour, 3 hours, 6hours or 12 hours; every day, every 2 days, 3 days or 7 days; or everymonth, 2 months, 3 months, 6 months or 12 months.

The administration of the pharmaceutical composition can be systemicadministration or local administration, and can be appropriatelyselected according to the target organ, tissue, or cell location. Localadministration is preferred for in vivo treatments because the vesiclescan be administered in a sufficient amount to the site (organ, tissue,or cells) to be effective in treatment, but have no influence on othertissues. However, if the vesicles are targeted to a specific organ,tissue, or cell-type (e.g., by virtue of incorporating in the vesicle aprotein or lipid that binds a specific molecule on the target organ,tissue, or cell), then systemic administration through, for example,intravenous injection or the like can be used. Blood flow willsystemically transport the vesicles, which will then contact the targetorgan, tissue, or cells.

In the case of administration by injection, the injection site may be asite where the vesicle can exert its functions and attain the purpose ofthe pharmaceutical composition. Examples of injection sites includeintravenous, intraarterial, intrahepatic, intramuscular, intraarticular,intramedullary, intraspinal, intraventricular, percutaneous,subcutaneous, intracutaneous, intraperitoneal, intranasal, intestinaland sublingual sites. In one embodiment, direct administration to theepididymis is preferred.

III. Compositions and Kits

In yet another aspect, disclosed herein are pharmaceutical compositionscomprising an vesicle comprising a small RNA molecule (sRNA). Inembodiments, the sRNA is selected from the group consisting of a siRNA,a miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNA fragment. Inthose embodiments where the sRNA is a tRNA fragment, the tRNA fragmentcan be selected from the group consisting of a tRNA-Gly-CCC fragment, atRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment,a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTGfragment.

In some embodiments, the pharmaceutical composition is a vaginal foam orgel.

In some embodiments, the vesicle is an exosome; in yet otherembodiments, the exosome is an epididymosome. In further embodiments,the epididymosome is selected from the group consisting of caputepididymosome, corpus epididymosome, and cauda epididymosome. In otherembodiments, the vesicle is a seminosome or a prostasome. In otherembodiments, the vesicle is a microvesicle.

In embodiments of this third aspect, the vesicle comprises aheterologous RNA. In further embodiments, the heterologous RNA comprisesa small RNA (sRNA). In yet further embodiments, the sRNA is selectedfrom the group consisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA,a U-RNA, and a tRNA fragment. In those embodiments where the sRNA is atRNA fragment, the tRNA fragment can be selected from the groupconsisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, atRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment,a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In otherembodiments, the vesicle comprises autologous RNA. In such embodiments,the vesicle comprises an sRNA that can be selected from the groupconsisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and atRNA fragment. In those embodiments where the sRNA is a tRNA fragment,the tRNA fragment can be selected from the group consisting of atRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment,a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTTfragment, and a tRNA-His-GTG fragment. In other embodiments, the vesiclecomprises an artificial (synthetic) RNA. In such embodiments, thevesicle comprises an sRNA that can be selected from the group consistingof a siRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNAfragment. In those embodiments where the sRNA is a tRNA fragment, thetRNA fragment can be selected from the group consisting of atRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment,a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTTfragment, and a tRNA-His-GTG fragment. In an embodiment, the vesiclecomprises a transgene.

Kits

In some embodiments, components necessary to perform the methodsdisclosed herein are included in kits. For example, vesicles can beformulated into pharmaceutical compositions and supplied in a vessel foruse as a vaginal foam or gel for in vivo use. In other embodiments,vesicles can be supplied in a vessel suspended in a buffer or media forin vitro or in vivo use, such as would be suitable for contactingisolated sperm with the vesicles. In some embodiments, the vesiclesincorporate heterologous molecular cargo. In some embodiments, thismolecular cargo is an RNA molecule (such as a sRNA) or a transgene.

Reagents included in kits can be supplied in containers of any sort suchthat the life of the different components are preserved and are notadsorbed or altered by the materials of the container. For example,sealed glass ampules may contain lyophilized components (such asvesicles), or buffers that have been packaged under a neutral,non-reacting gas, such as nitrogen. Suitable buffers include those thatmaintain the integrity of the vesicles over time. Ampules may consist ofany suitable material, such as glass, organic polymers (i.e.,polycarbonate, polystyrene, etc.), ceramic, metal or any other materialtypically employed to hold reagents. Other examples of suitablecontainers include simple bottles that may be fabricated from similarsubstances as ampules, and envelopes that may have foil-lined interiors,such as aluminum or alloy. Other containers include test tubes, vials,flasks, bottles, syringes, or the like. Containers may have a sterileaccess port, such as a bottle having a stopper that can be pierced by ahypodermic injection needle. Other containers may have two compartmentsthat are separated by a readily removable membrane that upon removalpermits the components to mix. Removable membranes may be glass,plastic, rubber, etc.

Kits can also be supplied with instructional materials. Instructions maybe printed on paper or other substrate and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, DVD,SD card, videotape, audio tape, etc. Detailed instructions may not bephysically associated with the kit; instead, a user may be directed toan internet web site specified by the manufacturer or distributor of thekit, or supplied as electronic mail.

EXAMPLES Example 1—Mouse Husbandry

Mice used in this study were primarily FVB/NJ strains, obtained fromJackson Laboratories (Bar Harbor, Me.). All animal care and useprocedures were in accordance with guidelines of the InstitutionalAnimal Care and Use Committee (University of Massachusetts). Animalswere raised on one of two diets—defined control diet (AIN-93G; Bioserv;Flemington, N.J.) or a Low Protein diet based on AIN-93g (10% of proteinrather than 19%, remaining mass made up with sucrose)—as previouslydescribed (Carone, B R, et al. 2010. Cell 143: 1084-1096). Because ithas been observed that in natural matings that paternal dietary effectsare substantially less penetrant when using females from our long termmouse colony, the experiments described herein have been restricted tothe use of female mice whose parents or grandparents had been obtainedfrom the animal vendor.

Example 2—Epididymosome Isolation

An adult male mouse (8-12 weeks old) was sacrificed using double killmethod (Isofluorane treatment followed by spinal dislocation). Next,cauda epididymis was dissected out and placed in a dish containing 1 mlWhitten's media (100 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄,5.5 mM Glucose, 1 mM Pyruvic acid, 4.8 mM Lactic acid (hemicalcium), andHEPES 20 mM) pre-warmed at 37° C. The epididymides were then gentlysqueezed using forceps to isolate the epididymal luminal content. Thedish was then placed in an incubator set at 37° C. with 5% CO₂ for 15minutes to allow any remaining epididymal content to release from thetissue. Next, the media containing epididymal luminal content wastransferred to a 1.5 ml tube and allowed to incubate for an additional15 minutes. At the end of the 15 minutes, any tissue pieces ornon-motile sperm settled down at the bottom of the tube and all thecontents of the tube except for the bottom approximately 50 μl weretransferred to a fresh tube. Next, the tube was spun in a tabletopcentrifuge at 2000×g for 5 minutes to pellet down sperm. Thesupernatant, which contained epididymosomes, was then transferred to afresh tube and centrifuged at 10000×g for 30 minutes at 4° C. to get ridof any non-sperm cells and cellular debris. Supernatant from this spinwas then transferred to a polycarbonate thick wall tube (13×56 mm,Beckman Coulter (Brea, Calif.), Catalog number 362305) and centrifugedat 120000×g for 2 hours at 4° C. in a table top ultracentrifuge (BeckmanOptima TL) using a TLA100.4 rotor. The pellet from this spin was thenwashed with 500 μl 1×PBS and centrifuged for another 2 hours at 120000×gat 4° C. Finally, the pellet containing epididymosomes was resuspendedin 50 μl ice-cold 1×PBS, transferred to a 1.5 ml tube, and flash frozenin liquid nitrogen.

Example 3—Small RNA Cloning

Total RNA was combined with an equal volume of Gel Loading Buffer II(Ambion; ThermoFisher Scientific; Carlsbad, Calif.), loaded onto a 15%Polyacrylamide with 7M Urea and 1×TBE gel, and run at 15 W in 1×TBEuntil the dye front was at the very bottom of the gel (approximately 25minutes for Criterion™ minigels (Bio-Rad; Hercules, Calif.)). Afterstaining with SYBR™ Gold (Life Technologies; Carlsbad, Calif.) for 7minutes, and destaining in 1×TBE for 7 minutes, gel slices correspondingto 18-40 nucleotides were then cut from the gel. Gel slices were thenground using a pipette tip or plastic pestle and mixed with 750 μl of0.3 M NaCl-TE, pH 7.5 prior to incubation with shaking on a thermomixerovernight at room temperature. The samples were then filtered using a0.4 μm cellulose acetate filter (Costar®; Corning; Corning, N.Y.) toremove gel debris. The eluate was transferred to a new low bindingmicrocentrifuge tube and 20 μg of glycoblue and 1 volume of isopropanol(approximately 700 μl) were added. Samples were precipitated for 30 ormore minutes at −20° C.

Size selection of the small RNAs was then followed by the ligation of a3′ adaptor and then a barcoded 5′ adaptor as described by Gu et al(2009, Mol. Cell 36: 231-244). The libraries were then converted to DNAusing SuperScript III® reverse transcriptase (Invitrogen; ThermoFisherScientific) and amplified by sequential rounds of PCR, to first addshort primer tails and then longer primer tails, providing the productswith the correct adaptor sequences for deep sequencing. Libraries werethen sequenced by Illumina HiSeq 2000 (Illumina; San Diego; CA) at theUniversity of Massachusetts Deep Sequencing Core (Worcester, Mass.).

Example 4—Normalization and Data Analysis

For each small RNA library, rRNA-mapping reads (which were highlyabundant in testis and epididymis samples, but rare in epididymosome andsperm samples) were removed. Remaining reads were mapped to murinetRNAs, to the unique sequences present in the 467 defined pachytenepiRNA clusters (Li, X Z, et al. 2013. Mol. Cell 50:67-81), toRepeatmasker (Institute for Systems Biology; Seattle, Wash.) (tRNAentries from Repeatmasker were deleted to avoid duplicating tRNA-mappingreads), to miRbase (Kozomara, A and S Griffiths-Jones, 2011. NAR 39(Database Issue): D152-D157) and to Refseq (Pruitt, K C D et al., 2014.NAR 42(1): D756-D763) (using RSEM (web link: deweylab.github.io/RSEM/)to separate distinct mRNA isoforms). Non rRNA-mapping reads werenormalized to parts per million mapped reads.

Example 5—ES Cell Culture and Transfection

E14 Embryonic Stem Cell (ESC) lines were grown in DMEM (Gibcom™;ThermoFisher Scientific), and transfections were carried out in inOpti-MEM™ (Gibco™; ThermoFisher Scientific) in 6 well plates (Fazzio, TG, et al. 2008. Cell 134: 162-174), with 9.5 cm² wells of ES cellsseeded at a density of 2.3×10⁵ cells/mL. One ng of antisense LNAcontaining oligonucleotides (synthesized by Exiqon; Woburn, Mass.) weretransfected using Lipofectamine™ 2000 (Invitrogen, ThermoFisherScientific) for 16 hours, then ESCs were allowed to recover for 32hours. Controls included Lipofectamine™ (Fisher Scientific) only (Mock)and anti-GFP shRNA transfections. RNA extraction was performed at theend of 48 hours using the standard TRIzol® (Ambion, Life Technologies;Carlsbad, Calif.) protocol. RNA extracted from mouse ES cells wasprepared for hybridization on Mouse GeneChip® 2.0 ST arrays (Affymetrix;Santa Clara, Calif.) using the GeneChip® WT PLUS kit from Affymetrix.

Example 6—In Vitro Fertilization, Embryo Culture, RNA Microinjection,and Single Embryo RNA-Seq

In vitro fertilization (IVF) was performed according to Nagy(Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., ed. 3, 2003)). FVB/NJ micewere used as egg donors and sperm was isolated from males fed dietaryregimes as previously described. Fertilization took place in 250 μl ofHTF media covered in mineral oil, pre-gassed in 5% CO₂ at 37° C.

IVF-derived control zygotes were placed in KSOM medium in 5% CO₂ 5% O₂incubator for 2 hours after IVF. Embryos were then washed twice in FHMmedium containing 0.1% PVA, and subject to micromanipulation. Embryoswere microinjected with either H3.3-GFP mRNA (control group) or H3.3-GFPmRNA+tRF-Gly-GCC antisense RNA (experimental group). RNA concentrationsused for microinjections were: 100 ng/μl for H3.3-GFP and 200 ng/μl fortRF-Gly-GCC antisense RNA. The sequence of the tRF-Gly-GCC antisense RNAis 5′GCG AGA AUU CUA CCA CUG AAC CAC CAA UGC 3′ (SEQ ID NO:1). After themicroinjections, embryos were placed back into culture, and GFPfluorescence was verified at the 2-cell stage. GFP-positive injectedembryos were cultured until the 4-cell stage, when they were collectedand processed for single-embryo RNA sequencing.

Single embryo RNA-Seq was carried out using the SMART-Seq protocol(Ramskold, S, et al. 2012. Nat. Biotechnol. 30: 777-782; Shalek, A K, etal. 2013. Nature 498: 236-240).

Example 7—Epididymal Delivery of Small RNAs to Immature Sperm

To interrogate the molecular mechanisms underlying transmission ofpaternal dietary information to offspring, the small (<40 nt) RNArepertoire of mouse sperm were characterized. Sperm were isolated fromthe cauda epididymis and subjected to several wash steps including adetergent wash with epithelial lysis buffer, yielding preparations thatwere routinely >99.5% pure as assessed by microscopy. Sperm RNA wasisolated, subjected to size selection (<40 nt), and small RNAs werecharacterized by cloning (with or without “healing” of 3′ ends by PNKtreatment) and deep sequencing as previously described (Gu, W. et al.2013. Cell, 151: 1488-1500). The resulting sequencing libraries show aremarkable abundance of approximately 28-32 nt tRNA fragments (tRFs) inmature sperm (approximately 80% of all small RNAs with cloneable 3′ends), as well as less abundant peaks of 19 nt and 22 nt RNAs (FIG. 1A).The tRFs in the dataset were derived from the 5′ ends of tRNAs (noevidence was found for persistence of 3′ fragments in mature sperm), anduntreated RNAs typically exhibit 2-4 predominant 3′ ends with a seriesof lower-abundance products potentially deriving from degradation oralternative cleavage/processing sites (FIGS. 1B-1D). As previouslyreported (Peng, H et al. 2012. Cell Res. 22:1609-1612), 5′ fragments oftRNA-Glu-CTC and tRNA-Gly-GCC were particularly abundant, although thepresently described dataset demonstrated that levels of 5′ tRFs derivedfrom other tRNA-glycine isoacceptors and from tRNA-valine isoacceptorswere comparable to those of tRF-Gly-GCC. 5′ tRFs were also highlyabundant in cauda sperm obtained from B. taurus, revealing thatextensive tRNA cleavage in gametes is conserved among mammals.

Example 8—tRNA Fragments are Abundant in the Epididymis

This example shows that tRNA fragments are abundant in the epididymis;the data are presented in FIG. 2. FIG. 2A shows a Northern blot analysisof total RNA isolated from testis, cauda epididymis, and caputepididymis, as indicated. Each panel (except the right-most panel,missing testis samples) shows paired samples in which each of the threetissues was obtained from the same animal. For each pair of samples, theleft sample was isolated from a Control animal and right sample wasisolated from a littermate consuming a Low Protein diet. Theapproximately 55-60 nt band observed in epididymis samples fortRNA-Gly-GCC and tRNA-Val-CAC varied somewhat in abundance betweensamples. This band almost certainly represents a T loop tRNA cleavageproduct rather than an intact tRNA differing in size from theapproximately 75 nt tRNA by virtue of amino acid charging, as (1) itmigrates at approximately 55-60 nt, which is too short to be an intacttRNA, (2) this band was observed both when using RNAs isolated underacidic conditions and using RNAs isolated under more basic deacylatingconditions (not shown), and (3) it is absent in testis samples. FIG. 2Bshows the quantitation of Northern blot data. For the indicated tRNAs,levels of the approximately 30 nt tRNA fragment were quantitated andnormalized to 5S RNA abundance. Bars show levels of tRFs in testis,caput epididymis, and cauda epididymis, normalized to testis levels.Error bars show s.e.m. FIG. 2C shows pie charts showing the percentageof small RNAs mapping to the indicated features, for each tissue.rRNA-mapping reads are excluded. Here, piRNAs refer to all small RNAsmapping either to Repeatmasker or to unique piRNA clusters (Li, X Z etal. 2013. Molecular Cell, 50: 67-81).

Example 9—tRNA Processing in the Epididymis

To explore the biogenesis of tRNA fragments found in sperm, small RNAsequencing data were generated for 18 testis samples from 10-12 week oldmales, and published data generated from testes of animals at varyingages after birth were reanalyzed (Li, X. Z., et al. 2013. MolecularCell, 50:67-81). As previously reported (Peng, H et al. 2012. Cell Res.22:1609-1612), very few small RNAs from testis mapped to tRNAs, with <8%of all <40 nt RNAs (excluding rRNA-mapping reads) mapping to tRNAs (FIG.3A). Northern blots against 5′ ends of tRNAs confirmed barely detectablelevels of tRNA cleavage products in testes. The spectrum of specifictRFs also differed between the testis, proximal caput epididymis, anddistal cauda epididymis. Similar results were obtained with small RNAprofiles of various testicular sperm fractions, including primaryspermatocytes, early and late round spermatids, and testicularspermatozoa (FIG. 4). In all four populations very low levels of tRNAfragments were observed, raising the question of where the tRNAfragments present in mature sperm might originate.

In a Northern blot analysis of tRNA cleavage, samples of the epididymiswere also included. The epididymis is the convoluted tubular structurein which sperm undergo post-testicular maturation over the course of 1-2weeks, moving from caput to corpus to cauda epididymis. Curiously,abundant 5′ tRFs were identified throughout the epididymis, but not intestes—for both tRNAs analyzed the approximately 30 nt 5′ tRNA fragmentthat was previously sequenced in sperm was observed (Peng, H et al.2012. Cell Res. 22:1609-1612) and FIG. 1). Levels of tRF-Gly-GCC weresimilar in the caput and cauda epididymis samples, while tRF-Val-CAC wasreproducibly more abundant in the cauda epididymis than in the caput.

Deep sequencing of small RNAs from caput and cauda epididymal samplesconfirmed high levels of 5′ tRFs in the epididymis. The overall tRFabundance increased from approximately 8% of all small RNA reads(excluding rRNA fragments) in testis to approximately 39% in the caputepididymis to approximately 64% in the cauda epididymis. Results fortRF-Glu-CTC, tRF-Gly-GCC, and tRF-Val-CAC were further validated inadditional samples by Taqman. Not only do overall tRF levels increasedramatically more distally in the male reproductive system, but thespectrum of specific tRFs differs between testis, caput epididymis, andcauda epididymis (FIGS. 5A and 5B). For instance, valine tRFs aregenerally more abundant distally (cauda>caput), whereas levels ofvarious glycine isoacceptor tRFs either peak in the caput epididymis orare high throughout the epididymis (consistent with the describedNorthern blotting results).

Example 10—Epididymosomes Carry Abundant tRFs that Match the Cauda SpermRNA Repertoire

The finding of robust tRNA cleavage in the epididymis, but not intestis, raises the surprising possibility that the abundant tRFs incauda sperm might originate from the epididymal epithelium rather thanduring testicular spermatogenesis. How might such trafficking from theepididymis to maturing sperm occur? During transit through theepididymis sperm gain scores of proteins (Dacheux, J L and F Dacheux.2013. Reproduction, 147: R27-R42; Sullivan, R, et al. 2013.Reproduction, 146: R21-R35) via fusion with small extracellular vesiclesknown as epididymosomes (Sullivan, R. et al. 2007. Asian J. Androl. 9:483-491; Sullivan, R, et al. 2013. Reproduction, 146: R21-R35). Becauseextracellular vesicles carry functional RNAs in multiple systems(Valadi, H, et al. 2007. Nat. Cell Biol. 9: 654-659; Regev-Rudzki, N, etal. 2013. Cell, 153: 1120-1133; Gibbings, D and O Voinnet. 2010. TrendsCell Biol. 20: 491-501), epididymosomes might be responsible for thedramatic alterations in the sperm RNA payload that occur duringepididymal transit.

Epididymosomes were purified from the cauda epididymis of 6-12 week oldmale mice by differential centrifugation. Purified epididymosomes weresomewhat heterogeneous in size, with a major size class centered aroundapproximately 150 nm (that occasionally revealed subpeaks ofapproximately 120 and approximately 170 nm), as well as a far lessabundant group of approximately 250-350 nm vesicles (FIGS. 6A and 6B).

Deep sequencing of small RNA libraries prepared from cauda epididymosomesamples (n=15) revealed several size classes of small RNAs, includinghighly abundant (approximately 87% of total reads) 28-34 nt tRNAfragments as well as lower levels of microRNAs and piRNAs (FIG. 7).RNaseA treatment of epididymosomes prior to RNA extraction had littleeffect on tRF abundance (FIG. 6C), consistent with these RNAs beingpresent in vesicles or otherwise protected, rather than free in theepididymal lumen. In addition, to ensure that the vesicles purified fromthe epididymis were not generated from maturing sperm, epididymosomeswere isolated from male tdrd1−/− mice (in which spermatogenesis isimpaired), confirming high levels of tRFs in these vesicles (FIG. 6C).

Small RNAs found in epididymosomes closely mirrored those found in caudasperm. For example, the most abundant RNA species in epididymosomes were5′ fragments of tRNA-Glu-CTC, followed by the 5′ ends oftRNA-Val-CAC/AAC and tRNA-Gly-GCC/CCC. Overall, the entire RNA payloadof mature sperm was remarkably well-correlated (r=0.98) with the RNAspresent in purified epididymosomes (FIG. 7). The small RNAs that wereenriched in sperm relative to epididymosomes included those mapping torepeat elements or unique piRNA clusters, as well as fragments of mRNAsinvolved in spermatogenesis (Prm1, for example). These RNAs thus almostcertainly reflect RNAs gained during spermatogenesis, but the remainingRNAs, which represented the vast majority of RNAs in cauda sperm, werefound at similar levels in sperm and in epididymosomes. Epididymosomescarried a highly similar RNA payload to that found in sperm, suggestingthat these vesicles may be responsible for delivering tRNA fragments andperhaps other small RNAs to maturing sperm.

Example 11—Vesicles Carrying tRNA Fragments Originate in the Epididymis

As fluid flow in the epididymis proceeds from testis through theepididymis and onwards to the vas deferens, luminal contents of thecauda epididymis could reflect a mixture of species secreted from avariety of upstream locations. In order to further investigate theorigin of tRF-containing vesicles in the reproductive tract,epididymosomes were purified from the caput epididymis (FIGS. 8A-8E).Purified caput epididymosomes had a similar size distribution to that ofcauda epididymosomes, although the modal size of cauda epididymosomeswas slightly larger than that of caput epididymosomes, and caudaepididymosomes also included larger (250-500 nm) particles. Small RNApopulations from caput epididymosomes (n=7) were isolated and subject todeep sequencing as above. Intriguingly, cauda and caput epididymosomesdiffered markedly in the relative abundance of microRNAs versus tRNAfragments. The relatively low abundance of tRFs in caput epididymosomesstrongly argues for an epididymal origin for the abundant tRFs in caudaepididymosomes, as any vesicles originating in the testis should ifanything be over-represented in the caput epididymosome samples relativeto the cauda.

Beyond these bulk changes in the abundance of general classes of smallRNAs, marked differences in the specific RNAs in each epididymosomepopulation were also observed. While not particularly abundant overall,mRNA fragments as a class were comparatively more abundant in caputepididymosomes, with the greatest enrichment for fragments of mRNAs thatare highly expressed in the caput epididymis (Johnston, D S. 2005. Biol.Reprod. 73: 404-413), such as Lcn5, Defb12, and Adam28. MicroRNAs wereoverall more abundant in caput epididymosomes, with individual microRNAsvarying in their relative enrichment in the two samples. tRNA fragmentsvaried considerably in relative abundance as well, with caudaepididymosomes gaining abundant tRF-Val-CAC, tRF-Val-AAC, andtRF-Gly-CCC while exhibiting relative “loss” of isoleucine and leucinetRFs. Overall, differences between caput and cauda epididymosomes insmall RNA abundance were moderately well-correlated to the analogousdifferences between epididymal epithelium samples, supporting thehypothesis that epididymosomes from a given luminal region likelyoriginate in the underlying epithelium. Together, these observationsstrongly support a model in which extracellular vesicles are secretedthroughout the male reproductive tract, with different sections of thetract releasing different RNA cargos.

Example 12—Epididymosomes Deliver Small RNAs to Sperm

The strong correlation between the small RNA cargo of epididymosomes andthat of cauda sperm, along with published evidence that epididymosomescan fuse with sperm and deliver other macromolecular cargo (Sullivan, R.et al. 2007. Asian J. Androl. 9: 483-491; Sullivan, R, et al. 2013.Reproduction, 146: R21-R35), suggests that epididymosomes areresponsible for shaping the RNA payload of maturing sperm. In order toisolate mature sperm that had not yet completed epididymal transit,sperm from the caput epididymis was purified and subjected them to smallRNA-Seq. Caput sperm (n=10) carried high levels of tRNA fragmentsindicating that the dramatic increase in tRNA fragment abundance insperm relative to testis occurs either very late during testicularspermatogenesis, or during the first approximately 3-5 days ofepididymal transit. That said, variation between caput and cauda spermsamples revealed extensive differences in the abundance of specificsmall RNAs. Examining tRNA fragment dynamics in detail, proximal-distalbiases for specific tRFs along the epididymis, and in epididymosomes,were also reflected in tRF dynamics in maturing sperm. Ratios of tRFs(as well as other small RNA classes) between caput and cauda sperm werewell-correlated with the caput/cauda ratios observed in epididymosomesand epididymis (FIG. 5). This does not result from artefactualcontamination of caput sperm samples with epididymosomes, as sequencingof small RNAs from caput sperm samples isolated with or without a stepof washing with detergent revealed that this washing protocol easilyremoved epididymosome-enriched RNAs.

In all three sample types analyzed—epididymis, epididymosomes, andsperm—key tRFs exhibited consistent biases in their enrichment along theproximal-distal axis of the epididymis. A small subset of tRFs wasgenerally enriched in the proximal epididymis, with most leucine andisoleucine isoacceptors generally being enriched in caput samples ofepididymis, epididymosomes, and sperm. In contrast, a dramatic apparentgain of tRF-Val-AAC/CAC between caput and cauda samples was observed,which was validated by Taqman in multiple independent samples. Thesedata support the hypothesis that fusion of caput sperm with caudaepididymosomes results in gain of tRF-Val-CAC and other RNAs, but couldalso be explained if small RNAs are globally degraded in sperm duringepididymal transit, with tRF-Val-CAC and related species being resistantto this degradation.

To determine whether epididymosomes can deliver their RNAs to caputsperm, caput sperm were stringently purified over Percoll gradients,incubated them with cauda epididymosomes at 37° C. for 1 or 2 hours,then pelleted and washed the “reconstituted” sperm (FIG. 9).Epididymosomal fusion with caput sperm was observed to be sufficient todeliver both tRF-Val-CAC and multiple other cauda-enriched tRFs to caputsperm (FIG. 9A), confirming that tRF-bearing epididymosomes either arecapable of fusing with sperm to deliver their small RNA cargo(Caballero, J N et al. 2013. PLOS ONES 8: e65364) or adhere to caputsperm strongly enough to resist removal by several consecutive washingsteps. These results were repeated using the more abundant caput spermsamples obtainable from the bull B. taurus, with cauda epididymosomefusion with caput sperm (n=4) resulting in delivery of tRF-Val-CAC andother tRFs to relatively immature caput sperm (FIG. 9B).

Taken together, these experiments are most consistent with a mechanismof RNA biogenesis in mammalian sperm in which tRFs generated in theepididymis are trafficked to sperm in epididymosomes.

Example 13—Functions of Sperm tRNA Fragments in Stem Cells and inEmbryos

Next, potential downstream targets of the small RNAs in sperm wereconsidered, initially using embryonic stem (ES) cells as an experimentalsystem amenable to mechanistic analysis. Here, the function of specifictRFs was interfered with using antisense LNA-containing oligonucleotidesin ES cell culture, and genome-wide analysis of RNA abundance (usingAffymetrix microarrays and RNA-Seq) was carried out to assay theconsequences of tRNA fragment inhibition. The majority of antisenseoligos had no effect on mRNA abundance, suggesting that the targetedtRFs are not functional in ES cells, or that they exerted regulatoryeffects that were not assayed by mRNA abundance.

In contrast, interfering with tRF-Gly-GCC function using anLNA-containing antisense oligonucleotide resulted in dramaticupregulation of approximately 50 genes, with several genes beingupregulated over 10-fold (FIGS. 10A-10C). Upregulation of these geneswas consistently observed by microarray in seven separate transfections,and further confirmed in four additional replicates by RNA-Seq. Thesegenes were unaffected by antisense LNA oligos directed against the 5′end of tRNA-Ser-GCT, the 5′ ends of other tRNA-Gly isoacceptors, oragainst the middle or the 3′ end of tRNA-Gly-GCC (FIG. 10B). This lastfinding strongly suggests that changes in gene expression caused byinterfering with the 5′ fragment of tRNA-Gly-GCC are unlikely to be anartifact of interfering with the function of the intact tRNA.Surprisingly, all the genes upregulated in tRF-Gly-GCC knockdowns arehighly expressed in 2-cell and 4-cell embryos, and have been shown to beregulated by the long terminal repeat (LTR) of an endogenousretroelement known as MERVL (Macfarlan, T S, et al. 2011. Genes Dev.25:594-607; Macfarlan, T S, et al. 2012. Nature 487: 57-63) (FIG. 10D).Transfection studies using ES cell lines carrying fluorescent reportersdriven by the LTR of MERVL revealed a modest increase (approximately25-40%) in the fraction of “MERVL positive” cells upon tRF-Gly-GCCinhibition, independently confirming the link between tRF-Gly-GCC andthe MERVL LTR.

To determine whether the effects of tRF-Gly-GCC inhibition observed intissue culture also hold in a more physiological context, zygotes (n=27)were microinjected with an antisense oligo directed against tRF-Gly-GCC.These embryos were then allowed to develop to the 4-cell stage andsubjected to single embryo RNA-Seq (Ramskold, D., et al. 2012. Nat.Biotechnol. 30: 777-782; Shalek, A K et al. 2013. Nature, 498: 236-240).Strikingly, significant upregulation of 72 transcripts in embryossubject to tRF-Gly-GCC inhibition was observed compared to controlembryos (n=28), with the majority of upregulated genes having previouslybeen identified as MERVL targets (Macfarlan, T S, et al. 2012. Nature487: 57-63) (FIGS. 10E and 10F).

Example 14—Paternal Dietary Effects on Preimplantation Development

This example shows that paternal diet effects preimplantationdevelopment. The data are shown in FIGS. 11 and 12. Given the robustconnection between a diet-regulated small RNA and a highly specific setof target genes, could tRF-Gly-GCC targets be affected inpreimplantation embryos generated using sperm from animals consumingControl or Low Protein diet? Single-embryo RNA-Seq (Ramskold, D., et al.2012. Nat. Biotechnol., 30: 777-782; Shalek, A K, et al. 2013. Nature,498: 236-240) of individual embryos cultured to various stages ofdevelopment robustly clustered embryos by developmental stage (FIGS.11A-B), with the first two principal components of the datasetrepresenting oocyte-derived transcripts (PC1), and embryonic genomeactivation (PC2).

As single embryo RNA-Seq data are not suitable for identification ofmodest changes in individual mRNAs, consistent changes in largergenesets were searched: the subset of MERVL targets that respond totRF-Gly-GCC inhibition (FIG. 10) and remaining MERVL targets (Macfarlan,T S, et al. 2012. Nature, 487: 57-63). At the 2-cell stage bothtRF-Gly-GCC targets and remaining MERVL targets were downregulated inLow Protein embryos relative to Control (FIG. 11C), consistent with thehypothesis that tRF-Gly-GCC in sperm affects expression of MERVL targetsin early embryos. Several independent tests of this hypothesis werecarried out. First, control zygotes were injected with <40 nt RNApopulations purified from Control and Low Protein sperm, finding thatLow Protein RNAs could inhibit tRF-Gly-GCC targets in 2-cell embryos(FIG. 11D) indicating that paternal diet can affect preimplantation generegulation via RNAs in sperm. Second, further defining the relevant RNAfrom Low Protein sperm, microinjection of a synthetic tRF-Gly-GCC oligoresulted in repression of MERVL target genes in 2-cell embryos (FIG.11E). Finally, as tRFs in sperm are gained during epididymal transit,embryos were generated via intracytoplasmic sperm injection (ICSI) usingtesticular spermatozoa or cauda sperm. Consistent with the higher levelsof tRF-Gly-GCC in cauda sperm, embryos generated using cauda spermexpressed MERVL targets at lower levels than embryos generated usingtesticular sperm (FIG. 11F). Together, these findings all support thehypothesis that tRF-Gly-GCC in sperm is capable of delaying orrepressing MERVL target expression in 2-cell embryos.

Lastly, tRF-Gly-GCC is observed to be one of several abundant RNAsregulated by Low Protein diet, and MERVL-driven genes are not the onlydiet-responsive genes in preimplantation embryos. Most notably,ribosomal protein genes (RPGs) were downregulated in Low Proteinembryos, and, correspondingly, Low Protein embryos develop slower thanControls (FIG. 12; discussed below) (Mitchel, M, et al. 2011. Fertil.Steril., 95: 1349-1353).

FIGS. 12A-12H show paternal dietary effects on preimplantationdevelopment. FIG. 12A shows subjected cumulative distribution plot forall genes encoding ribosomal protein genes. X axis shows the relativeexpression of these genes in Low Protein IVF embryos, compared toControl. Grey line shows distribution of dietary effects on all non-RPGgenes, for all four stages. Left shift at the 2-cell stage showsdownregulation of RPGs in Low Protein 2-cell embryos. FIGS. 12B-E showGSEA plots for various sets of genes involved in ribosome biogenesis atthe indicated developmental stages. FIG. 12F shows an example image of ablastocyst stained with DAPI and anti-Cdx2 to image total cell numberand trophectoderm cells. FIG. 12G shows that Low Protein dietreproducibly alters developmental tempo. FIG. 12H shows aggregated datafor three replicate experiments, showing the number of blastocysts withthe indicated number of cells, for embryos generated via IVF usingControl or Low Protein sperm, as indicated.

Example 15—Dietary Effects on tRNAs in Testes

This example shows that when the levels of intact tRNAs are assayed inthe testis, there is no correlation between dietary effects ontesticular tRNA levels and tRF changes in cauda sperm. The data areshown in FIG. 13. FIG. 13A shows a schematic illustrating assay for tRNAcharging analysis. RNA purified from a given tissue was isolated underacidic conditions to preserve charged tRNAs, and subjected to the threetreatments shown to enable deep sequencing characterization of charged,uncharged, and total tRNA levels. FIG. 13B shows validation of tRNAcharging protocol. Budding yeast grown in the presence (+HIS) or absence(−HIS) of histidine were subjected to the tRNA analysis shown in FIG.13A. Changes in tRNA abundance for charged and uncharged tRNAs are shownon the y axis, sorted by the change in charged tRNA abundance. Asexpected, charged tRNA-His levels dropped dramatically after two hoursof histidine starvation, while levels of uncharged tRNA-His increased.FIG. 13C shows testicular tRNA abundance correlation with codon bias inthe mouse. The x axis shows intact tRNA abundance in testis (total tRNAis shown here but similar results hold for uncharged or charged tRNAdatasets) in log scale, and the y axis shows the corresponding codonabundance (in codon frequency/1000) in all murine mRNAs, or in the 47most-highly expressed mRNAs in testis. Data for testis mRNA abundance isfrom Carone et al. (Carone, B R, et al. 2010. Cell 143: 1084-1096). FIG.13D shows validation of tRNA charging analysis. Scatterplot showsabundance of approximately 60-80 nt RNAs in the total RNA protocol (xaxis, log scale) compared to abundance of RNAs in the charged tRNAprotocol (y axis, log scale). While tRNA levels are broadly consistentbetween the two protocols (charged/uncharged ratios vary up toapproximately 10-fold between individual tRNAs), other RNAs captured inthe total RNA protocol, mostly snoRNAs (some of which are of similarsize to tRNAs), are approximately 20-100 fold less abundant in thecharged tRNA library. FIGS. 13E-G show Low Protein vs. Control effectson tRNA levels for total (FIG. 13E), uncharged (FIG. 13F), and charged(FIG. 13G) tRNA levels in testis. FIG. 13H shows that dietary effects onsperm tRFs are not explained by effects on intact tRNA abundance intestes. Log ratio between Control and Low Protein males is shown fortotal tRNA levels in testis (x axis) compared to tRNA fragment levels incauda sperm (y axis).

Example 16—Consistent Dietary Effects are Observed Throughout theReproductive Tract

This example shows that there are consistent dietary effects throughoutthe reproductive tract. The data are presented in FIG. 14. FIG. 14Ashows the dietary effects on small RNA abundance in testes and caput andcauda epididymis samples. Each heatmap shows log 2 of LowProtein/Control RNA abundance for a pair of samples, showing RNAs (rows)that exhibit consistent dietary effects across >75% of samples. FIG. 14Bshows the coherent dietary effects on tRF-Gly and let-7 family membersthroughout the male reproductive tract. For each RNA, bars show averageand standard error of the mean for Low Protein effects on the abundanceof the RNA species in the indicated tissue. Changes with a nominal pvalue of <0.05 (paired t test, not corrected for multiple testing) areindicated with asterisks.

Example 17—RNA Populations in Caput Sperm

In this example, the RNA populations in caput sperm are detailed,showing that the RNA payload of caput sperm differs substantially fromthat of cauda sperm. The data are shown in FIG. 15. FIG. 15A shows thatunwashed caput sperm are contaminated with RNAs abundant in caputepididymosomes. Caput sperm were isolated with and without washing withan epithelial cell lysis buffer. RNA isolated from unwashed caput spermincluded numerous microRNAs that were most abundant in caputepididymosomes. FIG. 15B shows a comparison of small RNA payloads ofcauda vs. caput sperm for all RNA species with an abundance of at least1 ppm in both sperm populations. These changes in RNA abundance couldresult from extant RNAs from caput sperm being degraded during furthertransit through the epididymis, or from small RNAs being gained viaprocessing or trafficking during post-testicular maturation. FIG. 15Cshows the proximal-distal biases observed for epididymis (x axis) wererecapitulated in cauda vs. caput sperm samples (y axis). For clarityonly RNAs are shown with at least 50 ppm abundance in at least one ofthe four sample types (cauda or caput, sperm or epididymis) are shown.The correlation coefficient for each RNA class is shown adjacent to itslabel. FIG. 15D shows that there is a gain in all four tRFs from caputto cauda. Data for the four tRFs indicated was normalized to let-7b, andhere the average cauda/caput difference for each tissue is shownplus/minus the standard deviation. Similar results were obtained usingmiR-21 as a normalized control. In addition, nearly-identical resultswere obtained using Taqman assays for the 23, 27, or 29 nt variants oftRF-Gly-GCC (only data for 27 nt is shown). These data are consistenteither with a general gain of tRFs from caput to cauda samples of allthree tissue types—epididymis, epididymosomes, and sperm—or loss oflet-7 or miR-21. FIG. 15E shows that tRF-Val-CAC is stronglycauda-enriched. Data from FIG. 15D are shown with tRF-Val-CAC normalizedto tRF-Glu-CTC rather than to microRNAs. Northern blots performedagainst the 5′end of tRNA-Gly-GCC for samples of bull caput sperm andbull cauda sperm, show that caput sperm carry intact tRNAs (FIG. 15F).

Example 18—Dietary Information is Carried in Sperm

This example shows that metabolic gene expression is altered inoffspring generated via in vitro fertilization (IVF) using spermobtained from animals consuming a control or low-protein diet. Despitethe potential for IVF and embryo culture to obscure paternal effects onoffspring metabolism, it was found that, compared with control IVFoffspring, IVF-derived offspring of males consuming a low-protein dietexhibited significant hepatic upregulation of the gene encoding thecholesterol biosynthesis enzyme squalene epoxidase (Sqle). These resultsare shown in FIG. 16.

FIGS. 16A-16C show that dietary information is carried in sperm. FIG.16A shows the sperm from males consuming Control or Low Protein dietwhich were used to fertilize oocytes gathered from Control females.Two-cell stage embryos were then implanted into pseudopregnant femalesand allowed to develop to birth. At 3 weeks of age, offspring weresacrificed (n=92 for Control, n=86 for Low Protein), and livers wereharvested for analysis of Sqle, a gene previously shown to beupregulated in offspring of Low Protein males relative to Control malesCarone et al., Cell 2010; 143: 1084-1096). Sqle levels (normalized toActb) are shown for all offspring as individual points, with horizontallines showing mean expression. FIG. 12B shows the cumulativedistribution of Sqle expression for all offspring generated usingControl or Low Protein sperm, as indicated.

FIG. 16C shows consistent litter effects. Here, Sqle levels wereaveraged for all offspring of a given litter. As sperm samples werealways obtained from male siblings split to different diets, litterpairs resulting from paired fathers were compared, with each dotrepresenting the ratio of Sqle expression between appropriately pairedlitters.

Example 19—Mechanistic Basis for tRF-Gly-GCC Regulation of MERVL Targets

This example provides data supporting the mechanistic basis fortRF-Gly-GCC regulation of MERVL targets. The data are shown in FIG. 17.FIGS. 17A-B show the mechanistic basis for tRF-Gly-GCC regulation ofMERVL. FIG. 17A shows RNA-Seq and ribosome footprinting data for Sp110.Genome browser view shows aggregated data for four independent replicateES cell transfections with shRNAs targeting GFP, and an antisense oligotargeting tRF-Gly-GCC. FIG. 17B shows that RNA abundance and ribosomefootprinting data are highly correlated. Scatterplot shows the effect oftRF-Gly-GCC inhibition, expressed as the log 2 of the median of the fourLNA transfections divided by the median of the eight control replicates(four mock, four GFP KD). Genes exhibiting a 2-fold difference betweenGFP KD and mock, and genes with maximum abundance of <2 FPKM in anyindividual replicate, were excluded from this scatterplot.

Example 20—Observations from Examples 1-19

The results from the previous examples show (1) that effects of paternaldiet on offspring are mediated via information found in sperm (FIG. 15),(2) that diet alters the level of small RNAs, including specific tRNAfragments, throughout the male reproductive tract and in mature sperm(FIGS. 1 and 7), and (3) that tRNA fragments can regulate expression oftranscripts driven by endogenous retroelements (FIGS. 8-9). The dataalso uncover the temporal dynamics of small RNA biogenesis duringpost-testicular maturation (FIGS. 2-4), and strongly suggest a role forepididymosomes in transmitting small RNAs from somatic cells of theepididymis to maturing gametes.

A role for epididymosomes in small RNA trafficking to sperm Perhaps themost surprising hypothesis raised from the results of these Examples isthat epididymosomes deliver a payload of small RNAs to maturing sperm.The idea that epididymal cells are partly responsible for the RNApayload of sperm is compelling given the increasing number of organismsin which gametogenesis involves a key role for small RNA communicationbetween germ cells and somatic support cells (Bourc'his, D and OVoinnet. 2010. Science, 330: 617-622). Four observations support thehypothesis. First, extremely low levels of tRNA fragments were found inthe murine testis, instead observing increasingly abundant tRFsthroughout the epididymis. Moreover, during epididymal transit, levelsof a number of tRFs increase in sperm between the proximal and distalsegments. Second, the small RNA payload of purified epididymosomes is aremarkable match for the small RNAs found in cauda sperm. In the veryunlikely case that sperm tRNA fragments do not originate in theepididymis, this observation would then either be an astonishingcoincidence if epididymosomal RNAs serve no regulatory function, or morelikely would hint at potential regulatory roles of epididymosomal RNAsin lumicrine signaling or signaling to the female reproductive tract(Bromfield, J J, et al. 2014. Proc. Natl. Acad. Sci. USA, 111:2200-2205; Vojitech, L, et al. 2014. Nucleic Acids Res., 42: 7290-7304).Third, fusion of purified epididymosomes with caput sperm in vitrodelivers tRNA fragments to the resulting “reconstituted” sperm,demonstrating that the epididymosomes bearing tRFs either can fuse withcaput sperm or very stably adhere to sperm. Finally, although the majorsmall RNAs (glycine tRFs and let-7) that respond to diet in mature spermare also diet-regulated in the testis (as well as the epididymis), otherdiet-responsive small RNAs in sperm only exhibit dietary responses inepididymis but not in testis.

Dietary Effects on Small RNAs in Mammalian Sperm

The key changes in small RNA observed in sperm of animals raised on LowProtein diet are observed throughout the male reproductive tract.Generally, at least five levels at which diet could exert effects on thelevels of a given tRF in sperm can be identified, by influencing: (1)intact tRNA abundance, either via transcription or stability, (2) tRNAcleavage, regulated potentially by tRNA charging status or by dietarysignaling to tRNA-modifying enzymes such as Dnmt2 or Nsun2, (3) tRFstability, (4) tRF sorting into epididymosomes, or (5) sperm fusion withepididymosomes—this category includes dietary regulation offusion-related cell surface proteins, but also mechanisms involvingchanges in sperm maturation time or epididymis luminal flow rate thatcould affect how long sperm spend in different parts of the epididymis.At present, dietary effects on tRF processing, stability, or traffickingappear to be the most likely scenario for at least a subset ofdiet-regulated tRFs.

tRF Regulation of an Endogenous Retroelement

How might diet-regulated small RNAs in sperm have the ability to impactthe phenotype of offspring? tRF-Gly-GCC was the focus of these studiesthanks to its readily apparent role in altering mRNA abundance in EScells—other abundant tRFs such as tRF-Gly-TCC may play roles inregulation of genes not expressed in ES cells, or may exert regulatoryeffects that are not apparent in mRNA abundance measures (e.g., ontranslation), and it will be interesting to determine whether theseother abundant tRFs in sperm have effects on preimplantationdevelopment. tRF-Gly-GCC is extremely unlikely to be uniquelyresponsible for the effects of paternal Low Protein diet on offspringcholesterol metabolism, as let-7 and many other small RNAs changeabundance in Low Protein sperm, and many more genes (such as RPGs)change in preimplantation embryos fertilized using these sperm than justMERVL target genes (FIG. 8E). Thus, we feel the likeliest scenario isthat paternal dietary effects are analogous to complex disease genetics,with multiple separate factors each contributing a fraction of thequantitative phenotype.

Inhibition of tRF-Gly-GCC, but not related tRFs, results in dramaticderepression in both ES cells and in early embryos of a subset(approximately 50 of approximately 500) of transcripts that areregulated by dispersed LTRs of the endogenous retroelement MERVL(Macfarlan, T S, et al. 2011. Genes Dev., 25: 594-607). Moreover,embryos generated using sperm from Low Protein males reveal significantchanges in MERVL target mRNA abundance (FIG. 10), consistent with theidea that tRFs delivered by sperm could affect gene regulation in theearly embryo. The mechanistic basis for the observed effects oftRF-Gly-GCC on repression of MERVL LTRs is of interest—although tRNAsnearly universally act as primers for retroelement replication,homology-driven tRF regulation of MERVL is unlikely here as (1) MERVLutilizes tRNA-Leu, not tRNA-Gly, to prime reverse transcriptase, (2)many of the genes in our dataset are associated with isolated LTRs andappear to lack the adjacent tRNA “primer binding sequence”, (3) primerbinding sequences for ERVs typically have homology to the 3′ end oftRNAs, not the 5′ end, and (4) transfection of the antisense LNA totRF-Gly-GCC does not affect levels of tRNA-Leu fragments in ES cells.Shown in these examples is that regulation of MERVL targets is unlikelyto be a secondary effect of altered translation of MERVL regulators bytRF-Gly-GCC, while reporter assays show that removing the MERVL LTR fromits genomic context—many of the tRF-Gly-GCC targets are found in largechromosomal regions with many MERVL LTRs nearby—does not completelyeliminate the ability of the LTR to respond to tRF-Gly-GCC inhibition.

The MERVL regulon provides an intriguing connection to offspringmetabolism. MERVL-driven genes are highly expressed in totipotent earlyembryos (Kigami, D, et al. 2003. Biol. Reprod., 68: 651-654), but asmall fraction of otherwise pluripotent embryonic stem cells alsoexpress the MERVL program, and MERVL positive cells are functionallytotipotent (Macfarlan, T S, et al. 2012. Nature, 487: 57-63). It is wellknown that alterations in placental function (as induced by uterineartery ligation or caloric restriction) lead to altered cholesterol andglucose metabolism in offspring (Rando, O J and Simmons, R A. 2015.Cell, 161: 93-105). It is hypothesized that tRF-Gly-GCC regulation ofthe MERVL program could alter the tempo of early development, or altercell fate allocation in the early embryo. While there was no significantdifference in the percentage of Cdx2-positive cells between Control andLow Protein embryos (73+/−5% vs. 71+/−7%), Low Protein embryosconsistently exhibited delayed growth relative to Control embryos.Interestingly, altered growth kinetics in early embryogenesis have beenshown to occur in response to paternal obesity, which also has beenlinked to offspring metabolism (McPherson, N O, et al. 2013. PLoS One,8(8)e71459).

Future studies will shed further light on the role of the epididymis insensing environmental conditions, on the mechanistic basis forregulation of RNA levels in sperm, and on effects of tRNA fragments onpreimplantation development and placentation.

Example 21—Caput Epididymosomes Deliver Small RNAs to TesticularSpermatozoa

The Examples described herein demonstrate that testicular spermatozoahave scarce levels of tRFs, and caput sperm are highly abundant in thesesmall RNAs. Epididymosomes secreted by the epithelium of caudaepididymis have been found to have similar RNA payload as that of themature sperm and can deliver small RNAs to the relatively “immature”caput sperm (see, e.g., Sharma et al., Science 2016; 351(6271):391-396).

To test whether tRFs and other small RNAs are delivered to testicularsperm upon entry into epididymis via fusion with epididymosomes presentin the caput epididymis, testicular spermatozoa were reconstituted byfusing them with caput epididymosomes (FIG. 18A). Testicular spermatozoawere incubated with caput epididymosomes for two hours and then purifiedby multiple washes to isolate “reconstituted” spermatozoa. Next, thelevels of specific small RNAs in reconstituted spermatozoa were examinedusing TaqMan qRT-PCR assays.

It was determined that tRFs, such as tRF-Glu-CTC and tRF-Val-CAC, whichare highly abundant in caput epididymosomes, were up-regulated more than2-fold in reconstituted spermatozoa compared to the mock fusions (FIG.18B). Deep sequencing of small RNAs from reconstituted spermatozoarevealed consistent results. Higher levels of tRFs and miRNAs wereobserved in reconstituted spermatozoa compared to mock controls (FIG.18A, 18C-18D).

Several lines of evidence prove that the small RNA content ofreconstituted sperm is altered due to epididymosome fusion/delivery ofsmall RNAs: 1) as piRNAs are not expressed in epididymis, there arescant levels of piRNAs in epididymosomes (Sharma et al., Science 2016;351(6271): 391-396), and no change in the levels of piRNAs was detectedin reconstituted spermatozoa (piRNAs are all on the diagonal axis of thescatter plot in FIG. 18D); 2) an increase in the levels of small RNA wasthe primary population of RNA detected, supporting that RNA is deliveredto sperm; and 3) reconstituted sperm showed an increase in miRNAs andtRFs that were specifically highly abundant in epididymosomes, such as,e.g., miR-10a/b, miR-148, miR-143, tRF-ValCAC, tRF-GluCTC, tRF-GlyGCCand tRF-HisGTG.

The reconstitution of testicular sperm recapitulated testicular sperm tocaput sperm maturation step in vitro. For instance, it was determinedthat the reconstituted sperm showed 10% higher levels of tRFs comparedto testicular spermatozoa (FIG. 18A). In addition, specific small RNAchanges were also very well recapitulated. Reconstituted sperm werefound to have a higher abundance of caput sperm-enriched microRNAs suchas miR-10a/b, miR-143, miR-141, and miR-200a. As such, caputepididymosomes were capable of fusing with mature testicular spermatozoato deliver their small-RNA cargo. Without intending to be bound byscientific theory, taken together these experiments are most consistentwith a mechanism of RNA biogenesis in mammalian sperm in which smallRNAs generated in the epididymis are trafficked to sperm inepididymosomes.

Example 22—Analysis of Gene Regulation Effects in Embryos Made Via ICSIUsing Caput Sperm, and Cauda Sperm

To determine the effects of epididymal maturation on phenotype in thefollowing generation, experiments are carried out in which zygotes aregenerated via intracytoplasmic sperm injection (ICSI) using spermobtained from the caput epididymis or from the cauda epididymis. Suchzygotes are then allowed to develop into 2-cell embryos, to blastocysts,or are implanted into females and carried to term. Gene regulation isstudied in the preimplantation embryos, and metabolic traits aremeasured in grown offspring, to identify the consequences of usingimmature sperm to fertilize oocytes.

Example 23—Microinjection of Other Small RNAs into Zygotes; Early GeneRegulation and Metabolic Sequelae Assays

To determine the effects of specific small RNAs on phenotype in thefollowing generation, experiments are carried out in which controlzygotes are injected with specific small RNAs, such as tRF-Val-CAC, andallowed to develop into 2-cell embryos, to blastocysts, or are implantedinto females and carried to term. Gene regulation is studied in thepreimplantation embryos, and metabolic traits are measured in grownoffspring, to identify the functions of specific small RNAs in earlydevelopment and future health.

The invention is not to be limited in scope by the specific embodimentsand examples described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

All references (e.g., publications or patents or patent applications)cited herein are incorporated herein by reference in their entiretiesand for all purposes to the same extent as if each individual reference(e.g., publication or patent or patent application) was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes. Other embodiments are within the following claims.

1-81. (canceled)
 82. A pharmaceutical composition comprising ansRNA-containing vesicle isolated from an epididymosome.
 83. Thepharmaceutical composition of claim 82, wherein the sRNA is selectedfrom the group consisting of a siRNA, a miRNA, a piRNA, a snoRNA, asrRNA, a U-RNA, and a tRNA fragment.
 84. The pharmaceutical compositionof claim 83, wherein the tRNA fragment is selected from the groupconsisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, atRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment,a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment.
 85. Thepharmaceutical composition of claim 82, wherein the pharmaceuticalcomposition is a vaginal foam or a gel.
 86. The pharmaceuticalcomposition of claim 82, wherein the epididymosome is selected from thegroup consisting of caput epididymosome, corpus epididymosome, and caudaepididymosome.
 87. The pharmaceutical composition of claim 82, whereinthe vesicle comprises a heterologous RNA.
 88. The pharmaceuticalcomposition of claim 87, wherein the heterologous RNA comprises a smallRNA (sRNA).
 89. The pharmaceutical composition of claim 88, wherein thesRNA is selected from the group consisting of a siRNA, miRNA, a piRNA, asnoRNA, a srRNA, a U-RNA, and a tRNA fragment.
 90. The pharmaceuticalcomposition of claim 89, wherein the sRNA is a tRNA fragment selectedfrom the group consisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCCfragment, a tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, atRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTGfragment.
 91. The pharmaceutical composition of claim 82, wherein thevesicle comprises autologous RNA.
 92. The pharmaceutical composition ofclaim 91, wherein the sRNA is selected from the group consisting of asiRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNA fragment.93. The pharmaceutical composition of claim 92, wherein the sRNA is atRNA fragment selected from the group consisting of a tRNA-Gly-CCCfragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, atRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment,and a tRNA-His-GTG fragment.
 94. The pharmaceutical composition of claim82, wherein the vesicle comprises an artificial or a synthetic RNA. 95.The pharmaceutical composition of claim 94, wherein the sRNA is selectedfrom the group consisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA,a U-RNA, and a tRNA fragment.
 96. The pharmaceutical composition ofclaim 95, wherein the sRNA is a tRNA fragment selected from the groupconsisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, atRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment,a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment.
 97. Thepharmaceutical composition of claim 82, wherein the vesicle comprises atransgene. 98-141. (canceled)